Transthyretin (TTR) iRNA compositions and methods of use thereof for treating or preventing TTR-associated diseases

ABSTRACT

The present invention provides iRNA agents, e.g., double stranded iRNA agents, that target the transthyretin (TTR) gene and methods of using such iRNA agents for treating or preventing TTR-associated diseases.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/221,651, filed Jul. 28, 2016, now U.S. Pat. No. 10,208,307, issued onFeb. 19, 2019, which claims the benefit of priority to U.S. ProvisionalPatent Application No. 62/199,563, filed on Jul. 31, 2015, and to U.S.Provisional Patent Application No. 62/287,518, filed on Jan. 27, 2016.The entire contents of each of the foregoing applications are herebyincorporated herein by reference.

This application is related to U.S. Provisional Patent Application No.61/881,257, filed Sep. 23, 2013, and International Application No.PCT/US2014/056923, filed Sep. 23, 2014, the entire contents of each ofwhich are hereby incorporated herein by reference. In addition, thisapplication is related to U.S. Provisional Application No. 61/561,710,filed on Nov. 18, 2011, International Application No. PCT/US2012/065601,filed on Nov. 16, 2012, U.S. Provisional Application No. 61/615,618,filed on Mar. 26, 2012, U.S. Provisional Application No. 61/680,098,filed on Aug. 6, 2012, U.S. application Ser. No. 14/358,972, filed onMay 16, 2014, no U.S. Pat. No. 9,399,775, issued on Jul. 26, 2016, U.S.application Ser. No. 15/188,317, filed on Jun. 21, 2016, U.S.application Ser. No. 16/738,014, filed on Jan. 9, 2020, andInternational Application No. PCT/US2012/065691, filed Nov. 16, 2012,the entire contents of each of which are hereby incorporated herein byreference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 18, 2018, isnamed 121301_03004_SeqListing.txt and is 68,327 bytes in size.

BACKGROUND OF THE INVENTION

Transthyretin (TTR) (also known as prealbumin) is found in serum andcerebrospinal fluid (CSF). TTR transports retinol-binding protein (RBP)and thyroxine (T4) and also acts as a carrier of retinol (vitamin A)through its association with RBP in the blood and the CSF. Transthyretinis named for its transport of thyroxine and retinol. TTR also functionsas a protease and can cleave proteins including apoA-I (the major HDLapolipoprotein), amyloid β-peptide, and neuropeptide Y. See Liz, M. A.et al. (2010) IUBMB Life, 62(6):429-435.

TTR is a tetramer of four identical 127-amino acid subunits (monomers)that are rich in beta sheet structure. Each monomer has two 4-strandedbeta sheets and the shape of a prolate ellipsoid. Antiparallelbeta-sheet interactions link monomers into dimers. A short loop fromeach monomer forms the main dimer-dimer interaction. These two pairs ofloops separate the opposed, convex beta-sheets of the dimers to form aninternal channel.

The liver is the major site of TTR expression. Other significant sitesof expression include the choroid plexus, retina (particularly theretinal pigment epithelium) and pancreas.

Transthyretin is one of at least 27 distinct types of proteins that is aprecursor protein in the formation of amyloid fibrils. See Guan, J. etal. (Nov. 4, 2011) Current perspectives on cardiac amyloidosis, Am JPhysiol Heart Circ Physiol, doi:10.1152/ajpheart.00815.2011.Extracellular deposition of amyloid fibrils in organs and tissues is thehallmark of amyloidosis. Amyloid fibrils are composed of misfoldedprotein aggregates, which may result from either excess production of orspecific mutations in precursor proteins. The amyloidogenic potential ofTTR may be related to its extensive beta sheet structure; X-raycrystallographic studies indicate that certain amyloidogenic mutationsdestabilize the tetrameric structure of the protein. See, e.g., SaraivaM. J. M. (2002) Expert Reviews in Molecular Medicine, 4(12):1-11.

Amyloidosis is a general term for the group of amyloid diseases that arecharacterized by amyloid deposits. Amyloid diseases are classified basedon their precursor protein; for example, the name starts with “A” foramyloid and is followed by an abbreviation of the precursor protein,e.g., ATTR for amyloidogenic transthyretin. Ibid.

There are numerous TTR-associated diseases, most of which are amyloiddiseases. Normal-sequence TTR is associated with cardiac amyloidosis inpeople who are elderly and is termed senile systemic amyloidosis (SSA)(also called senile cardiac amyloidosis (SCA) or cardiac amyloidosis).SSA often is accompanied by microscopic deposits in many other organs.TTR amyloidosis manifests in various forms. When the peripheral nervoussystem is affected more prominently, the disease is termed familialamyloidotic polyneuropathy (FAP). When the heart is primarily involvedbut the nervous system is not, the disease is called familialamyloidotic cardiomyopathy (FAC). A third major type of TTR amyloidosisis leptomeningeal amyloidosis, also known as leptomeningeal ormeningocerebrovascular amyloidosis, central nervous system (CNS)amyloidosis, or amyloidosis VII form. Mutations in TTR may also causeamyloidotic vitreous opacities, carpal tunnel syndrome, and euthyroidhyperthyroxinemia, which is a non-amyloidotic disease thought to besecondary to an increased association of thyroxine with TTR due to amutant TTR molecule with increased affinity for thyroxine. See, e.g.,Moses et al. (1982) J. Clin. Invest., 86, 2025-2033.

Abnormal amyloidogenic proteins may be either inherited or acquiredthrough somatic mutations. Guan, J. et al. (Nov. 4, 2011) Currentperspectives on cardiac amyloidosis, Am J Physiol Heart Circ Physiol,doi:10.1152/ajpheart.00815.2011. Transthyretin associated ATTR is themost frequent form of hereditary systemic amyloidosis. Lobato, L. (2003)J. Nephrol., 16:438-442. TTR mutations accelerate the process of TTRamyloid formation and are the most important risk factor for thedevelopment of ATTR. More than 85 amyloidogenic TTR variants are knownto cause systemic familial amyloidosis. TTR mutations usually give riseto systemic amyloid deposition, with particular involvement of theperipheral nervous system, although some mutations are associated withcardiomyopathy or vitreous opacities. Ibid.

The V30M mutation is the most prevalent TTR mutation. See, e.g., Lobato,L. (2003) J Nephrol, 16:438-442. The V122I mutation is carried by 3.9%of the African American population and is the most common cause of FAC.Jacobson, D. R. et al. (1997) N. Engl. J. Med. 336 (7): 466-73. It isestimated that SSA affects more than 25% of the population over age 80.Westermark, P. et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87 (7):2843-5.

Accordingly, there is a need in the art for effective treatments forTTR-associated diseases.

SUMMARY OF THE INVENTION

The present invention provides RNAi agents, e.g., double stranded RNAiagents, and compositions targeting the Transthyretin (TTR) gene. Thepresent invention also provides methods of inhibiting expression of TTRand methods of treating or preventing a TTR-associated disease in asubject using the RNAi agents, e.g., double stranded RNAi agents, of theinvention. The present invention is based, at least in part, on thediscovery that RNAi agents in which substantially all of the nucleotideson the sense strand and substantially all of the nucleotides of theantisense strand are modified nucleotides and that comprise no more than8 2′-fluoro modifications on the sense strand, no more than 6 2′-fluoromodifications on the antisense strand, two phosphorothioate linkages atthe 5′-end of the sense strand, two phosphorothioate linkages at the5′-end of the antisense strand, and a ligand, e.g., a GalNAc₃ ligand,are shown herein to be effective in silencing the activity of the TTRgene. These agents show surprisingly enhanced TTR gene silencingactivity. Without intending to be limited by theory, it is believed thata combination or sub-combination of the foregoing modifications and thespecific target sites in these RNAi agents confer to the RNAi agents ofthe invention improved efficacy, stability, potency, and durability.

Accordingly, in one aspect, the present invention provides doublestranded ribonucleic acid (RNAi) agents for inhibiting expression oftransthyretin (TTR) in a cell, wherein the RNAi agent comprises a sensestrand complementary to an antisense strand, wherein the antisensestrand comprises a region complementary to SEQ ID NO:2(5′-UGGGAUUUCAUGUAACCAAGA-3′), wherein each strand is about 14 to about30 nucleotides in length, wherein substantially all of the nucleotidesof the sense strand and substantially all of the nucleotides of theantisense strand are modified nucleotides, wherein the sense strandcomprises no more than 8 2′-fluoro modifications; wherein the antisensestrand comprises no more than 6 2′-fluoro modifications; wherein thesense strand and the antisense strand each independently comprise twophosphorothioate linkages at the 5′-terminus; and wherein the sensestrand is conjugated to at least one ligand.

In one embodiment, and wherein the double stranded RNAi agent isrepresented by formula (IIIe):

(IIIe) sense: 5′-N_(a)-Y Y Y-N_(b)-3′ antisense:3′-n_(p)′-N_(a)′-Y′Y′Y′-N_(b)′-5′

wherein:

n_(p)′ is a 2 nucleotide overhang and each nucleotide within n_(p)′ islinked to a neighboring nucleotide via a phosphorothioate linkage;

each N_(a), N_(b), N_(a)′ and N_(b)′ independently represents anoligonucleotide sequence comprising 0-25 nucleotides which are eithermodified or unmodified or combinations thereof, each sequence comprisingat least two differently modified nucleotides;

YYY and Y′Y′Y′ each independently represent one motif of three identicalmodifications on three consecutive nucleotides.

In one embodiment, the YYY motif occurs at or near the cleavage site ofthe sense strand. In one embodiment, the Y′Y′Y′ motif occurs at the 11,12 and 13 positions of the antisense strand from the 5′-end.

In one embodiment, the Y nucleotides contain a 2′-fluoro modification.

In one embodiment, the Y′ nucleotides contain a 2′-O-methylmodification.

The double stranded region may be 15-30 nucleotide pairs in length,17-23 nucleotide pairs in length, 17-25 nucleotide pairs in length,23-27 nucleotide pairs in length, 19-21 nucleotide pairs in length, or21-23 nucleotide pairs in length.

Each strand of the double stranded RNAi agent may have 15-30 nucleotidesor 19-30 nucleotides.

In one embodiment, the modifications on the nucleotides are selectedfrom the group consisting of a deoxy-nucleotide, a 3′-terminaldeoxy-thymine (dT) nucleotide, a 2′-O-methyl modified nucleotide, a2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a lockednucleotide, an unlocked nucleotide, a conformationally restrictednucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a2′-amino-modified nucleotide, a 2′-O-allyl-modified nucleotide,2′-C-alkyl-modified nucleotide, 2′-hydroxyl-modified nucleotide, a2′-methoxyethyl modified nucleotide, a 2′-O-alkyl-modified nucleotide, amorpholino nucleotide, a phosphoramidate, a non-natural base comprisingnucleotide, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitolmodified nucleotide, a cyclohexenyl modified nucleotide, a nucleotidecomprising a phosphorothioate group, a nucleotide comprising amethylphosphonate group, a nucleotide comprising a 5′-phosphate, and anucleotide comprising a 5′-phosphate mimic, and combinations thereof.

In one embodiment, the modifications on the nucleotides are 2′-O-methylor 2′-fluoro modifications.

The sense strand may comprise no more than 7 2′-fluoro modifications, nomore than 6 2′-fluoro modifications, no more than 5 2′-fluoromodification, no more than 4 2′-fluoro modifications, no more than 32′-fluoro modifications, or no more than 2 2′-fluoro modifications.

The antisense strand may comprise no more than 5 2′-fluoromodifications, no more than 4 2′-fluoro modifications, no more than 32′-fluoro modifications, or no more than 2 2′-fluoro modifications.

In one embodiment, the double stranded RNAi agent further comprises a5′-phosphate or a 5′-phosphate mimic at the 5′ nucleotide of theantisense strand. In another embodiment, the double stranded RNAi agentfurther comprises a 5′-phosphate mimic at the 5′ nucleotide of theantisense strand.

In one embodiment, the 5′-phosphate mimic is a 5′-vinyl phosphate(5′-VP).

In one embodiment, the ligand is one or more GalNAc derivatives attachedthrough a bivalent or trivalent branched linker. In another embodiment,the ligand is

In one embodiment, the ligand is attached to the 3′ end of the sensestrand.

In one embodiment, the double stranded RNAi agent is conjugated to theligand as shown in the following schematic

wherein X is O or S.

In one embodiment, the antisense strands comprise a nucleotide sequenceselected from the group consisting of5′-usCfsuugguuacaugAfaaucccasusc-3′ (SEQ ID NO: 6),5′-usCfsuugGfuuAfcaugAfaAfucccasusc-3′ (SEQ ID NO:7),5′-UfsCfsuugGfuuAfcaugAfaAfucccasusc-3′ (SEQ ID NO: 8), and5′-VPusCfsuugGfuuAfcaugAfaAfucccasusc-3′ (SEQ ID NO: 9), wherein a, c,g, and u are 2′-O-methyl (2′-OMe) A, C, G, or U; Af, Cf, Gf, and Uf are2′-fluoro A, C, G, or U; and s is a phosphorothioate linkage; and VP isa 5′-phosphate mimic.

In one embodiment, the sense and antisense strands comprise nucleotidesequences selected from the group consisting of5′-usgsggauUfuCfAfUfguaaccaaga-3′ (SEQ ID NO: 10) and5′-usCfsuugguuacaugAfaaucccasusc-3′ (SEQ ID NO: 6);5′-usgsggauUfuCfAfUfguaaccaaga-3′ (SEQ ID NO: 10) and5′-usCfsuugGfuuAfcaugAfaAfucccasusc-3′ (SEQ ID NO: 7);5′-usgsggauUfuCfAfUfguaaccaaga-3′ (SEQ ID NO: 10) and5′-UfsCfsuugGfuuAfcaugAfaAfucccasusc-3′ (SEQ ID NO: 8); and5′-usgsggauUfuCfAfUfguaaccaaga-3′ (SEQ ID NO: 10) and5′-VPusCfsuugGfuuAfcaugAfaAfucccasusc-3′ (SEQ ID NO: 9), wherein a, c,g, and u are 2′-O-methyl (2′-OMe) A, C, G, or U; Af, Cf, Gf, and Uf are2′-fluoro A, C, G, or U; and s is a phosphorothioate linkage; and VP isa 5′-phosphate mimic. In another embodiment, the sense and antisensestrands comprise the nucleotide sequences5′-usgsggauUfuCfAfUfguaaccaaga-3′ (SEQ ID NO: 10) and5′-usCfsuugGfuuAfcaugAfaAfucccasusc-3′ (SEQ ID NO: 7), wherein a, c, g,and u are 2′-O-methyl (2′-OMe) A, C, G, or U; Af, Cf, Gf, and Uf are2′-fluoro A, C, G, or U; and s is a phosphorothioate linkage. In yetanother embodiment, the RNAi agent is selected from the group of any oneof the RNAi agents listed in any one of Tables 1 and 3. In yet anotherembodiment, the RNAi agent is AD-65492.

In one aspect, the present invention provides double strandedribonucleic acid (RNAi) agents for inhibiting expression oftransthyretin (TTR) in a cell, wherein the RNAi agent comprises a sensestrand complementary to an antisense strand, wherein the antisensestrand comprises a region fully complementary to SEQ ID NO:2(5′-UGGGAUUUCAUGUAACCAAGA-3′), wherein each strand is about 14 to about30 nucleotides in length, wherein substantially all of the nucleotidesof the sense strand and substantially all of the nucleotides of theantisense strand are modified nucleotides, wherein the sense strandcomprises no more than 8 2′-fluoro modifications; wherein the antisensestrand comprises no more than 6 2′-fluoro modifications; wherein thesense strand and the antisense strand each independently comprise twophosphorothioate linkages at the 5′-terminus; and wherein the sensestrand is conjugated to at least one ligand, wherein the ligand is oneor more GalNAc derivatives attached through a bivalent or trivalentbranched linker.

In one embodiment, the double stranded RNAi agent is represented byformula (IIIe):

(IIIe) sense: 5′-N_(a)-Y Y Y-N_(b)-3′ antisense:3′-n_(p)′-N_(a)′-Y′Y′Y′-N_(b)′-5′

wherein:

n_(p)′ is a 2 nucleotide overhang and each nucleotide within n_(p)′ islinked to a neighboring nucleotide via a phosphorothioate linkage;

each N_(a), N_(b), N_(a)′ and N_(b)′ independently represents anoligonucleotide sequence comprising 8-10 nucleotides which are eithermodified or unmodified or combinations thereof, each sequence comprisingat least two differently modified nucleotides;

YYY and Y′Y′Y′ each independently represent one motif of three identicalmodifications on three consecutive nucleotides, and wherein themodifications are 2′-O-methyl or 2′-fluoro modifications.

The present invention also provides cells containing the double strandedRNAi agents of the invention, cells comprising the vectors of theinvention, and pharmaceutical compositions comprising the doublestranded RNAi agents of the invention or the vectors of the invention.

In one embodiment, the double stranded RNAi agent is administered in anunbuffered solution, e.g., saline or water.

In another embodiment, the double stranded RNAi agent is administeredwith a buffer solution. In one embodiment, the buffer solution comprisesacetate, citrate, prolamine, carbonate, or phosphate or any combinationthereof. In another embodiment, the buffer solution is phosphatebuffered saline (PBS).

In another aspect, the present invention provides methods of inhibitingtransthyretin (TTR) expression in a cell. The methods include (a)contacting the cell with the double stranded RNAi agents of theinvention, the vectors of the invention, or the pharmaceuticalcompositions of the invention; and (b) maintaining the cell produced instep (a) for a time sufficient to obtain degradation of the mRNAtranscript of a TTR gene, thereby inhibiting expression of the TTR genein the cell.

In one embodiment, the cell is within a subject.

In one embodiment, the subject is a human.

In one embodiment, the subject suffers from TTR-associated disease.

In one embodiment, the TTR expression is inhibited by at least about10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%,about 60%, about 70%, about 80%, about 90%, about 95%, about 98% orabout 100%.

In yet another aspect, the present invention provides methods oftreating a subject having a transthyretin (TTR)-associated disorder, byadministering to the subject a therapeutically effective amount of thedouble stranded RNAi agents of the invention, or the vectors of theinvention, or the pharmaceutical compositions of the invention, therebytreating the subject.

In yet another aspect, the present invention provides methods ofprophylactically treating a subject at risk of developing atransthyretin (TTR)-associated disorder, by administering to the subjecta prophylactically effective amount of the double stranded RNAi agentsof the invention, or the vectors of the invention, or the pharmaceuticalcompositions of the invention, thereby prophylactically treating thesubject.

In a further aspect, the present invention provides methods of treatinga subject having a transthyretin (TTR)-associated disorder. The methodsinclude administering to the subject a therapeutically effective amountof a double stranded RNAi agent, wherein the double stranded RNAi agentcomprises a sense strand complementary to an antisense strand, whereinthe antisense strand comprises a region complementary to SEQ ID NO:2(5′-UGGGAUUUCAUGUAACCAAGA-3′), wherein each strand is about 14 to about30 nucleotides in length, wherein substantially all of the nucleotidesof the sense strand and substantially all of the nucleotides of theantisense strand are modified nucleotides, wherein the sense strandcomprises no more than 8 2′-fluoro modifications; wherein the antisensestrand comprises no more than 6 2′-fluoro modifications; wherein thesense strand and the antisense strand each independently comprise twophosphorothioate linkages at the 5′-terminus; and wherein the sensestrand is conjugated to at least one ligand.

In a further aspect, the present invention provides methods ofprophylactically treating a subject at risk of developing atransthyretin (TTR)-associated disorder. The methods includeadministering to the subject a prophylactically effective amount of adouble stranded RNAi agent, wherein the double stranded RNAi agentcomprises a sense strand complementary to an antisense strand, whereinthe antisense strand comprises a region complementary to SEQ ID NO:2(5′-UGGGAUUUCAUGUAACCAAGA-3′), wherein each strand is about 14 to about30 nucleotides in length, wherein substantially all of the nucleotidesof the sense strand and substantially all of the nucleotides of theantisense strand are modified nucleotides, wherein the sense strandcomprises no more than 8 2′-fluoro modifications; wherein the antisensestrand comprises no more than 6 2′-fluoro modifications; wherein thesense strand and the antisense strand each independently comprise twophosphorothioate linkages at the 5′-terminus; and wherein the sensestrand is conjugated to at least one ligand.

In one aspect, the present invention provides methods of reducing,slowing, or arresting a Neuropathy Impairment Score (NIS) or a modifiedNIS (mNIS+7) in a subject having a transthyretin (TTR)-associateddisorder. The methods include administering to the subject atherapeutically effective amount of the double stranded RNAi agents ofthe invention, or the vectors of the invention, or the pharmaceuticalcompositions of the invention, thereby reducing, slowing, or arresting aNeuropathy Impairment Score (NIS) or a modified NIS (mNIS+7) in thesubject.

In a further aspect, the present invention provides methods of reducing,slowing, or arresting a Neuropathy Impairment Score (NIS) or a modifiedNIS (mNIS+7) a subject having a transthyretin (TTR)-associated disorder.The methods include administering to the subject a therapeuticallyeffective amount of a double stranded RNAi agent, wherein the doublestranded RNAi agent comprises a sense strand complementary to anantisense strand, wherein said antisense strand comprises a regioncomplementary to SEQ ID NO:2, wherein each strand is about 14 to about30 nucleotides in length, wherein substantially all of the nucleotidesof the sense strand and substantially all of the nucleotides of theantisense strand are modified nucleotides, wherein the sense strandcomprises no more than 8 2′-fluoro modifications; wherein the antisensestrand comprises no more than 6 2′-fluoro modifications; wherein thesense strand and the antisense strand each independently comprise twophosphorothioate linkages at the 5′-terminus; and wherein the sensestrand is conjugated to at least one ligand.

In one aspect, the present invention provides methods of increasing a6-minute walk test (6MWT) in a subject having a transthyretin(TTR)-associated disorder. The methods include administering to thesubject a therapeutically effective amount of the the double strandedRNAi agents of the invention, or the vectors of the invention, or thepharmaceutical compositions of the invention, thereby increasing a6-minute walk test (6MWT) in the subject.

In a further aspect, the present invention provides methods ofincreasing a 6-minute walk test (6MWT) in a subject having atransthyretin (TTR)-associated disorder. The methods includeadministering to the subject a therapeutically effective amount of adouble stranded RNAi agent, wherein the double stranded RNAi agentcomprises a sense strand complementary to an antisense strand, whereinsaid antisense strand comprises a region complementary to SEQ ID NO:2,wherein each strand is about 14 to about 30 nucleotides in length,wherein substantially all of the nucleotides of the sense strand andsubstantially all of the nucleotides of the antisense strand aremodified nucleotides, wherein the sense strand comprises no more than 82′-fluoro modifications; wherein the antisense strand comprises no morethan 6 2′-fluoro modifications; wherein the sense strand and theantisense strand each independently comprise two phosphorothioatelinkages at the 5′-terminus; and wherein the sense strand is conjugatedto at least one ligand.

In one embodiment, the double stranded RNAi agent is represented byformula (IIIe):

(IIIe) sense: 5′-N_(a)-Y Y Y-N_(b)-3′ antisense:3′-n_(p)′-N_(a)′-Y′Y′Y′-N_(b)′-5′

wherein:

n_(p)′ is a 2 nucleotide overhang and each nucleotide within n_(p)′ islinked to a neighboring nucleotide via a phosphorothioate linkage;

each N_(a), N_(b), N_(b) and N_(b)′ independently represents anoligonucleotide sequence comprising 0-25 nucleotides which are eithermodified or unmodified or combinations thereof, each sequence comprisingat least two differently modified nucleotides;

YYY and Y′Y′Y′ each independently represent one motif of three identicalmodifications on three consecutive nucleotides.

In one embodiment, the subject is a human.

In one embodiment, the subject is a subject suffering from aTTR-associated disease. In another embodiment, the subject is a subjectat risk for developing a TTR-associated disease. In one embodiment, thesubject at risk of developing a TTR-associated disease carries a TTRgene mutation that is associated with the development of a TTRassociated disease, or a subject with a family history of TTR-associateddisease, or a subject who has signs or symptoms suggesting thedevelopment of TTR amyloidosis.

In one embodiment, the TTR-associated disease is selected from the groupconsisting of senile systemic amyloidosis (SSA), systemic familialamyloidosis, familial amyloidotic polyneuropathy (FAP), familialamyloidotic cardiomyopathy (FAC), leptomeningeal/Central Nervous System(CNS) amyloidosis, and hyperthyroxinemia.

In one embodiment, the subject has a TTR-associated amyloidosis and themethod reduces an amyloid TTR deposit in the subject.

In one embodiment, the double stranded RNAi agent is administered to thesubject by an administration means selected from the group consisting ofsubcutaneous, intravenous, intramuscular, intrabronchial, intrapleural,intraperitoneal, intraarterial, lymphatic, cerebrospinal, and anycombinations thereof. In another embodiment, the double stranded RNAiagent is administered to the subject via subcutaneous, intramuscular orintravenous administration. In yet another embodiment, the doublestranded RNAi agent is administered to the subject via subcutaneousadministration.

In one embodiment, the methods further comprise assessing the level ofTTR mRNA expression or TTR protein expression in a sample derived fromthe subject.

In one embodiment, administering the double stranded RNAi agent does notresult in an inflammatory response in the subject as assessed based onthe level of a cytokine or chemokine selected from the group consistingof G-CSF, IFN-γ, IL-10, IL-12 (p70), IL1β, IL-1ra, IL-6, IL-8, IP-10,MCP-1, MIP-1α, MIP-1β, TNFα, and any combinations thereof, in a samplefrom the subject.

In one aspect, the present invention provides methods of treating asubject having a transthyretin (TTR)-associated disorder. The methodsinclude administering to the subject a fixed dose of about 12.5 mg toabout 200 mg (e.g., about 12.5 mg, about 25 mg, about 50 mg, about 75mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, or about 200mg) of a double stranded RNAi agent, wherein the double stranded RNAiagent comprises a sense strand complementary to an antisense strand,wherein the antisense strand comprises a region complementary to SEQ IDNO:2 (5′-UGGGAUUUCAUGUAACCAAGA-3′), wherein each strand is about 14 toabout 30 nucleotides in length, wherein substantially all of thenucleotides of the sense strand and substantially all of the nucleotidesof the antisense strand are modified nucleotides, wherein the sensestrand comprises no more than 8 2′-fluoro modifications; wherein theantisense strand comprises no more than 6 2′-fluoro modifications;wherein the sense strand and the antisense strand each independentlycomprise two phosphorothioate linkages at the 5′-terminus; and whereinthe sense strand is conjugated to at least one ligand.

In another aspect, the present invention provides methods ofprophylactically treating a subject at risk of developing atransthyretin (TTR)-associated disorder. The methods includeadministering to the subject a fixed dose of about 12.5 mg to about 200mg (e.g., about 12.5 mg, about 25 mg, about 50 mg, about 75 mg, about100 mg, about 125 mg, about 150 mg, about 175 mg, or about 200 mg) of adouble stranded RNAi agent, wherein the double stranded RNAi agentcomprises a sense strand complementary to an antisense strand, whereinthe antisense strand comprises a region complementary to SEQ ID NO:2(5′-UGGGAUUUCAUGUAACCAAGA-3′), wherein each strand is about 14 to about30 nucleotides in length, wherein substantially all of the nucleotidesof the sense strand and substantially all of the nucleotides of theantisense strand are modified nucleotides, wherein the sense strandcomprises no more than 8 2′-fluoro modifications; wherein the antisensestrand comprises no more than 6 2′-fluoro modifications; wherein thesense strand and the antisense strand each independently comprise twophosphorothioate linkages at the 5′-terminus; and wherein the sensestrand is conjugated to at least one ligand.

In one aspect, the present invention provides methods of treating asubject having a transthyretin (TTR)-associated disorder. The methodsinclude administering to the subject a dose of about 0.15 mg/kg to about2.5 mg/kg (e.g., about 0.15 mg/kg, about 0.3 mg/kg, about 0.6 mg/kg,about 1 mg/kg, about 1.25 mg/kg, about 2 mg/kg, about 2.5 mg/kg, orabout 3 mg/kg) of a double stranded RNAi agent, wherein the doublestranded RNAi agent comprises a sense strand complementary to anantisense strand, wherein the antisense strand comprises a regioncomplementary to SEQ ID NO:2 (5′-UGGGAUUUCAUGUAACCAAGA-3′), wherein eachstrand is about 14 to about 30 nucleotides in length, whereinsubstantially all of the nucleotides of the sense strand andsubstantially all of the nucleotides of the antisense strand aremodified nucleotides, wherein the sense strand comprises no more than 82′-fluoro modifications; wherein the antisense strand comprises no morethan 6 2′-fluoro modifications; wherein the sense strand and theantisense strand each independently comprise two phosphorothioatelinkages at the 5′-terminus; and wherein the sense strand is conjugatedto at least one ligand.

In one aspect, the present invention provides methods ofprophylactically treating a subject at risk of developing atransthyretin (TTR)-associated disorder. The methods includeadministering to the subject a dose of about 0.15 mg/kg to about 2.5mg/kg (e.g., about 0.15 mg/kg, about 0.3 mg/kg, about 0.6 mg/kg, about 1mg/kg, about 1.25 mg/kg, about 2 mg/kg, about 2.5 mg/kg, or about 3mg/kg) of a double stranded RNAi agent, wherein the double stranded RNAiagent comprises a sense strand complementary to an antisense strand,wherein the antisense strand comprises a region complementary to SEQ IDNO:2 (5′-UGGGAUUUCAUGUAACCAAGA-3′), wherein each strand is about 14 toabout 30 nucleotides in length, wherein substantially all of thenucleotides of the sense strand and substantially all of the nucleotidesof the antisense strand are modified nucleotides, wherein the sensestrand comprises no more than 8 2′-fluoro modifications; wherein theantisense strand comprises no more than 6 2′-fluoro modifications;wherein the sense strand and the antisense strand each independentlycomprise two phosphorothioate linkages at the 5′-terminus; and whereinthe sense strand is conjugated to at least one ligand.

In another aspect, the present invention provides methods of reducing,slowing, or arresting a Neuropathy Impairment Score (NIS) or a modifiedNIS (mNIS+7) in a subject having a transthyretin (TTR)-associateddisorder. The methods include administering to the subject a dose ofabout 0.15 mg/kg to about 2.5 mg/kg (e.g., about 0.15 mg/kg, about 0.3mg/kg, about 0.6 mg/kg, about 1 mg/kg, about 1.25 mg/kg, about 2 mg/kg,about 2.5 mg/kg, or about 3 mg/kg) of a double stranded RNAi agent,wherein the double stranded RNAi agent comprises a sense strandcomplementary to an antisense strand, wherein the antisense strandcomprises a region complementary to SEQ ID NO:2(5′-UGGGAUUUCAUGUAACCAAGA-3′), wherein each strand is about 14 to about30 nucleotides in length, wherein substantially all of the nucleotidesof the sense strand and substantially all of the nucleotides of theantisense strand are modified nucleotides, wherein the sense strandcomprises no more than 8 2′-fluoro modifications; wherein the antisensestrand comprises no more than 6 2′-fluoro modifications; wherein thesense strand and the antisense strand each independently comprise twophosphorothioate linkages at the 5′-terminus; and wherein the sensestrand is conjugated to at least one ligand.

In yet another aspect, the present invention provides methods ofincreasing a 6-minute walk test (6MWT) in a subject having atransthyretin (TTR)-associated disorder. The methods includeadministering to the subject a dose of about 0.15 mg/kg to about 2.5mg/kg (e.g., about 0.15 mg/kg, about 0.3 mg/kg, about 0.6 mg/kg, about 1mg/kg, about 1.25 mg/kg, about 2 mg/kg, about 2.5 mg/kg, or about 3mg/kg) of a double stranded RNAi agent, wherein the double stranded RNAiagent comprises a sense strand complementary to an antisense strand,wherein the antisense strand comprises a region complementary to SEQ IDNO:2 (5′-UGGGAUUUCAUGUAACCAAGA-3′), wherein each strand is about 14 toabout 30 nucleotides in length, wherein substantially all of thenucleotides of the sense strand and substantially all of the nucleotidesof the antisense strand are modified nucleotides, wherein the sensestrand comprises no more than 8 2′-fluoro modifications; wherein theantisense strand comprises no more than 6 2′-fluoro modifications;wherein the sense strand and the antisense strand each independentlycomprise two phosphorothioate linkages at the 5′-terminus; and whereinthe sense strand is conjugated to at least one ligand.

In one aspect, the present invention provides methods of treating asubject having a transthyretin (TTR)-associated disorder. The methodsinclude administering to the subject a fixed dose of about 10 mg toabout 600 mg, about 25 mg to about 500 mg, about 50 mg to about 500 mg,or about 80 mg to about 500 mg, about 25 mg to about 300 mg, about 50 mgto about 300 mg, or about 80 mg to about 300 mg (e.g., about 10, about20, about 30, about 40, about 50, about 60, about 70, about 75, about80, about 90, about 100, about 110, about 120, about 125, about 130,about 140, about 150, about 160, about 170, about 175, about 180, about190, about 200, about 210, about 220, about 225, about 230, about 240,about 250, about 260, about 270, about 275, about 280, about 290, about300, about 310, about 320, about 325, about 330, about 340, about 350,about 360, about 370, about 375, about 380, about 390, about 400, about410, about 420, about 425, about 430, about 440, about 450, about 460,about 470, about 475, about 480, about 490, about 500, about 510, about520, about 525, about 530, about 540, about 550, about 560, about 570,about 575, about 580, about 590, or about 600 mg) of a double strandedRNAi agent, wherein the double stranded RNAi agent comprises a sensestrand complementary to an antisense strand, wherein the antisensestrand comprises a region complementary to SEQ ID NO:2(5′-UGGGAUUUCAUGUAACCAAGA-3′), wherein each strand is about 14 to about30 nucleotides in length, wherein substantially all of the nucleotidesof the sense strand and substantially all of the nucleotides of theantisense strand are modified nucleotides, wherein the sense strandcomprises no more than 8 2′-fluoro modifications; wherein the antisensestrand comprises no more than 6 2′-fluoro modifications; wherein thesense strand and the antisense strand each independently comprise twophosphorothioate linkages at the 5′-terminus; and wherein the sensestrand is conjugated to at least one ligand.

In one aspect, the present invention provides methods ofprophylactically treating a subject at risk of developing atransthyretin (TTR)-associated disorder. The methods includeadministering to the subject a fixed dose of about 10 mg to about 600mg, about 25 mg to about 500 mg, about 50 mg to about 500 mg, or about80 mg to about 500 mg, about 25 mg to about 300 mg, about 50 mg to about300 mg, or about 80 mg to about 300 mg (e.g., about 10, about 20, about30, about 40, about 50, about 60, about 70, about 75, about 80, about90, about 100, about 110, about 120, about 125, about 130, about 140,about 150, about 160, about 170, about 175, about 180, about 190, about200, about 210, about 220, about 225, about 230, about 240, about 250,about 260, about 270, about 275, about 280, about 290, about 300, about310, about 320, about 325, about 330, about 340, about 350, about 360,about 370, about 375, about 380, about 390, about 400, about 410, about420, about 425, about 430, about 440, about 450, about 460, about 470,about 475, about 480, about 490, about 500, about 510, about 520, about525, about 530, about 540, about 550, about 560, about 570, about 575,about 580, about 590, or about 600 mg) of a double stranded RNAi agent,wherein the double stranded RNAi agent comprises a sense strandcomplementary to an antisense strand, wherein the antisense strandcomprises a region complementary to SEQ ID NO:2(5′-UGGGAUUUCAUGUAACCAAGA-3′), wherein each strand is about 14 to about30 nucleotides in length, wherein substantially all of the nucleotidesof the sense strand and substantially all of the nucleotides of theantisense strand are modified nucleotides, wherein the sense strandcomprises no more than 8 2′-fluoro modifications; wherein the antisensestrand comprises no more than 6 2′-fluoro modifications; wherein thesense strand and the antisense strand each independently comprise twophosphorothioate linkages at the 5′-terminus; and wherein the sensestrand is conjugated to at least one ligand.

In another aspect, the present invention provides methods of reducing,slowing, or arresting a Neuropathy Impairment Score (NIS) or a modifiedNIS (mNIS+7) in a subject having a transthyretin (TTR)-associateddisorder. The methods include administering to the subject a fixed doseof about 10 mg to about 600 mg, about 25 mg to about 500 mg, about 50 mgto about 500 mg, or about 80 mg to about 500 mg, about 25 mg to about300 mg, about 50 mg to about 300 mg, or about 80 mg to about 300 mg(e.g., about 10, about 20, about 30, about 40, about 50, about 60, about70, about 75, about 80, about 90, about 100, about 110, about 120, about125, about 130, about 140, about 150, about 160, about 170, about 175,about 180, about 190, about 200, about 210, about 220, about 225, about230, about 240, about 250 mg, about 260, about 270, about 275, about280, about 290, about 300, about 310, about 320, about 325, about 330,about 340, about 350, about 360, about 370, about 375, about 380, about390, about 400, about 410, about 420, about 425, about 430, about 440,about 450 mg, about 460, about 470, about 475, about 480, about 490,about 500, about 510, about 520, about 525, about 530, about 540, about550, about 560, about 570, about 575, about 580, about 590, or about 600mg) of a double stranded RNAi agent, wherein the double stranded RNAiagent comprises a sense strand complementary to an antisense strand,wherein the antisense strand comprises a region complementary to SEQ IDNO:2 (5′-UGGGAUUUCAUGUAACCAAGA-3′), wherein each strand is about 14 toabout 30 nucleotides in length, wherein substantially all of thenucleotides of the sense strand and substantially all of the nucleotidesof the antisense strand are modified nucleotides, wherein the sensestrand comprises no more than 8 2′-fluoro modifications; wherein theantisense strand comprises no more than 6 2′-fluoro modifications;wherein the sense strand and the antisense strand each independentlycomprise two phosphorothioate linkages at the 5′-terminus; and whereinthe sense strand is conjugated to at least one ligand.

In yet another aspect, the present invention provides methods ofincreasing a 6-minute walk test (6MWT) in a subject having atransthyretin (TTR)-associated disorder. The methods includeadministering to the subject affixed dose of about 10 mg to about 600mg, about 25 mg to about 500 mg, about 50 mg to about 500 mg, or about80 mg to about 500 mg, about 25 mg to about 300 mg, about 50 mg to about300 mg, or about 80 mg to about 300 mg (e.g., about 10, about 20, about30, about 40, about 50, about 60, about 70, about 75, about 80, about90, about 100, about 110, about 120, about 125, about 130, about 140,about 150, about 160, about 170, about 175, about 180, about 190, about200, about 210, about 220, about 225, about 230, about 240, about 250mg, about 260, about 270, about 275, about 280, about 290, about 300,about 310, about 320, about 325, about 330, about 340, about 350, about360, about 370, about 375, about 380, about 390, about 400, about 410,about 420, about 425, about 430, about 440, about 450 mg, about 460,about 470, about 475, about 480, about 490, about 500, about 510, about520, about 525, about 530, about 540, about 550, about 560, about 570,about 575, about 580, about 590, or about 600 mg) of a double strandedRNAi agent, wherein the double stranded RNAi agent comprises a sensestrand complementary to an antisense strand, wherein the antisensestrand comprises a region complementary to SEQ ID NO:2(5′-UGGGAUUUCAUGUAACCAAGA-3′), wherein each strand is about 14 to about30 nucleotides in length, wherein substantially all of the nucleotidesof the sense strand and substantially all of the nucleotides of theantisense strand are modified nucleotides, wherein the sense strandcomprises no more than 8 2′-fluoro modifications; wherein the antisensestrand comprises no more than 6 2′-fluoro modifications; wherein thesense strand and the antisense strand each independently comprise twophosphorothioate linkages at the 5′-terminus; and wherein the sensestrand is conjugated to at least one ligand.

In one embodiment, the double stranded RNAi agent is represented byformula (IIIe):

(IIIe) sense: 5′-N_(a)-Y Y Y-N_(b)-3′ antisense:3′-n_(p)′-N_(a)′-Y′Y′Y′-N_(b)′-5′

wherein:

n_(p)′ is a 2 nucleotide overhang and each nucleotide within n_(p)′ islinked to a neighboring nucleotide via a phosphorothioate linkage;

each N_(a), N_(b), N_(b) and N_(b)′ independently represents anoligonucleotide sequence comprising 0-25 nucleotides which are eithermodified or unmodified or combinations thereof, each sequence comprisingat least two differently modified nucleotides;

YYY and Y′Y′Y′ each independently represent one motif of three identicalmodifications on three consecutive nucleotides.

In one embodiment, the antisense strand comprises a nucleotide sequenceselected from the group consisting of5′-usCfsuugguuacaugAfaaucccasusc-3′ (SEQ ID NO: 6),5′-usCfsuugGfuuAfcaugAfaAfucccasusc-3′(SEQ ID NO: 7),5′-UfsCfsuugGfuuAfcaugAfaAfucccasusc-3′ (SEQ ID NO: 8), and5′-VPusCfsuugGfuuAfcaugAfaAfucccasusc-3′ (SEQ ID NO: 9), wherein a, c,g, and u are 2′-O-methyl (2′-OMe) A, C, G, or U; Af, Cf, Gf, and Uf are2′-fluoro A, C, G, or U; s is a phosphorothioate linkage; and VP is a5′-phosphate mimic.

In one embodiment, the sense and antisense strands comprise nucleotidesequences selected from the group consisting of5′-usgsggauUfuCfAfUfguaaccaaga-3′ (SEQ ID NO: 10) and5′-usCfsuugguuacaugAfaaucccasusc-3′ (SEQ ID NO: 6);5′-usgsggauUfuCfAfUfguaaccaaga-3′ (SEQ ID NO: 10) and5′-usCfsuugGfuuAfcaugAfaAfucccasusc-3′ (SEQ ID NO: 7);5′-usgsggauUfuCfAfUfguaaccaaga-3′ (SEQ ID NO: 10) and5′-UfsCfsuugGfuuAfcaugAfaAfucccasusc-3′(SEQ ID NO: 8); and5′-usgsggauUfuCfAfUfguaaccaaga-3′ (SEQ ID NO: 10) and5′-VPusCfsuugGfuuAfcaugAfaAfucccasusc-3′ (SEQ ID NO: 9), wherein a, c,g, and u are 2′-O-methyl (2′-OMe) A, C, G, or U; Af, Cf, Gf, and Uf are2′-fluoro A, C, G, or U; and s is a phosphorothioate linkage; and VP isa 5′-phosphate mimic.

In one embodiment, the sense and antisense strands comprise thenucleotide sequences

(SEQ ID NO: 10) 5′-usgsggauUfuCfAfUfguaaccaaga-3′ and (SEQ ID NO: 7)5′-usCfsuugGfuuAfcaugAfaAfucccasusc-3′,

wherein a, c, g, and u are 2′-O-methyl (2′-OMe) A, C, G, or U; Af, Cf,Gf, and Uf are 2′-fluoro A, C, G, or U; and s is a phosphorothioatelinkage.

The fixed dose of the double stranded RNAi agent may be administered tothe subject once about every 4 weeks, every 5 weeks, every six weeks,every eight weeks or quarterly.

The dose of the double stranded RNAi agent may be administered to thesubject once about every 4 weeks, every 5 weeks, every six weeks, everyeight weeks or quarterly.

In one embodiment, the double stranded RNAi agent is administered to thesubject about once every quarter.

In one embodiment, the double stranded RNAi agent is chronicallyadministered to the subject.

In one embodiment, the subject is a human.

In one embodiment, the subject is a subject suffering from aTTR-associated disease. In another embodiment, the subject is a subjectat risk for developing a TTR-associated disease. In one embodiment, thesubject at risk of developing a TTR-associated disease carries a TTRgene mutation that is associated with the development of a TTRassociated disease, or a subject with a family history of TTR-associateddisease, or a subject who has signs or symptoms suggesting thedevelopment of TTR amyloidosis.

In one embodiment, the TTR-associated disease is selected from the groupconsisting of senile systemic amyloidosis (SSA), systemic familialamyloidosis, familial amyloidotic polyneuropathy (FAP), familialamyloidotic cardiomyopathy (FAC), leptomeningeal/Central Nervous System(CNS) amyloidosis, and hyperthyroxinemia.

In one embodiment, the subject has a TTR-associated amyloidosis and themethod reduces an amyloid TTR deposit in the subject.

In one embodiment, the double stranded RNAi agent is administered to thesubject by an administration means selected from the group consisting ofsubcutaneous, intravenous, intramuscular, intrabronchial, intrapleural,intraperitoneal, intraarterial, lymphatic, cerebrospinal, and anycombinations thereof. In another embodiment, the double stranded RNAiagent is administered to the subject via subcutaneous, intramuscular orintravenous administration. In yet another embodiment, the doublestranded RNAi agent is administered to the subject via subcutaneousadministration, e.g., via self administration, e.g., via a pre-filledsyringe or auto-injector syringe.

In one embodiment, the methods further comprise assessing the level ofTTR mRNA expression or TTR protein expression in a sample derived fromthe subject.

In one embodiment, administering the double stranded RNAi agent does notresult in an inflammatory response in the subject as assessed based onthe level of a cytokine or chemokine selected from the group consistingof G-CSF, IFN-γ, IL-10, IL-12 (p70), IL1β, IL-1ra, IL-6, IL-8, IP-10,MCP-1, MIP-1α, MIP-1β, TNFα, and any combinations thereof, in a samplefrom the subject.

In one embodiment, the agent suitable for use in the methods of theinvention is AD-65492. AD-65492 may be chronically administered to thesubject every 4 weeks, every 5 weeks, or every six weeks, or everyquarter.

In one aspect, the present invention provides double strandedribonucleic acid (RNAi) agents for use in inhibiting expression oftransthyretin (TTR) in a cell. The agents include a sense strandcomplementary to an antisense strand, wherein the sense and antisensestrands comprise nucleotide sequences selected from the group consistingof any of the nucleotide sequences in Table 5.

In another aspect, the present invention provides double strandedribonucleic acid (RNAi) agents for use in inhibiting expression oftransthyretin (TTR) in a cell. The agents include a sense strandcomplementary to an antisense strand, the antisense strand comprising aregion of complementarity which comprises at least 15 contiguousnucleotides differing no more than 3 nucleotides from any one of theantisense sequences in Table 6, wherein substantially all of thenucleotides of the sense strand and substantially all of the nucleotidesof the antisense strand are modified nucleotides; and wherein the sensestrand is conjugated to at least one ligand.

The sense and antisense strand may comprise nucleotide sequencesselected from the group consisting of any of the nucleotide sequences inTable 6 or Table 7.

In one aspect the present invention provides double stranded ribonucleicacid (RNAi) agents for use in inhibiting expression of transthyretin(TTR) in a cell, wherein the RNAi agents comprise a sense strandcomplementary to an antisense strand, wherein the sense strand comprisesthe nucleotide sequence 5′-usgsggauUfuCfAfUfguaaccaaga-3′ (SEQ ID NO:10) and the antisense strand comprises the nucleotide sequence5′-usCfsuugGfuuAfcaugAfaAfucccasusc-3′ (SEQ ID NO: 7), wherein a, c, g,and u are 2′-O-methyl (2′-OMe) A, C, G, or U; Af, Cf, Gf, and Uf are2′-fluoro A, C, G, or U; and s is a phosphorothioate linkage.

In another aspect, the present invention provides methods of treating asubject suffering from a TTR-associated disease. The methods includeadministering to the subject a dose of about 50 mg to about 300 mg of adouble stranded RNAi agent, wherein the RNAi agent comprises a sensestrand complementary to an antisense strand, wherein the sense strandcomprises the nucleotide sequence 5′-usgsggauUfuCfAfUfguaaccaaga-3′ (SEQID NO: 10) and the antisense strand comprises the nucleotide sequence5′-usCfsuugGfuuAfcaugAfaAfucccasusc-3′ (SEQ ID NO: 7), wherein a, c, g,and u are 2′-O-methyl (2′-OMe) A, C, G, or U; Af, Cf, Gf, and Uf are2′-fluoro A, C, G, or U; and s is a phosphorothioate linkage, therebytreating the subject suffering from a TTR-associated disease.

In yet another aspect, the present invention provides methods ofprophylactically treating a subject at risk for developing aTTR-associated disease. The methods include administering to the subjecta dose of about 50 mg to about 300 mg of a double stranded RNAi agent,wherein the RNAi agent comprises a sense strand complementary to anantisense strand, wherein the sense strand comprises the nucleotidesequence 5′-usgsggauUfuCfAfUfguaaccaaga-3′ (SEQ ID NO: 10) and theantisense strand comprises the nucleotide sequence5′-usCfsuugGfuuAfcaugAfaAfucccasusc-3′ (SEQ ID NO: 7), wherein a, c, g,and u are 2′-O-methyl (2′-OMe) A, C, G, or U; Af, Cf, Gf, and Uf are2′-fluoro A, C, G, or U; and s is a phosphorothioate linkage, therebyprophylactically treating the subject at risk for developing aTTR-associated disease.

In one aspect, the present invention provides methods of reducing,slowing, or arresting a Neuropathy Impairment Score (NIS) or a modifiedNIS (mNIS+7) in a subject suffering from a TTR-associated disease or atrisk for developing a TTR-associated disease. The methods includeadministering to the subject a dose of about 50 mg to about 300 mg of adouble stranded RNAi agent, wherein the RNAi agent comprises a sensestrand complementary to an antisense strand, wherein the sense strandcomprises the nucleotide sequence 5′-usgsggauUfuCfAfUfguaaccaaga-3′ (SEQID NO: 10) and the antisense strand comprises the nucleotide sequence5′-usCfsuugGfuuAfcaugAfaAfucccasusc-3′ (SEQ ID NO: 7), wherein a, c, g,and u are 2′-O-methyl (2′-OMe) A, C, G, or U; Af, Cf, Gf, and Uf are2′-fluoro A, C, G, or U; and s is a phosphorothioate linkage, therebyreducing, slowing, or arresting a Neuropathy Impairment Score (NIS) or amodified NIS (mNIS+7) in the subject.

In another aspect, the present invention provides methods of increasinga 6-minute walk test (6MWT) in a subject suffering from a TTR-associateddisease or at risk for developing a TTR-associated disease. The methodsinclude administering to the subject a dose of about 50 mg to about 300mg of of a double stranded RNAi agent, wherein the RNAi agent comprisesa sense strand complementary to an antisense strand, wherein the sensestrand comprises the nucleotide sequence5′-usgsggauUfuCfAfUfguaaccaaga-3′ (SEQ ID NO: 10) and the antisensestrand comprises the nucleotide sequence5′-usCfsuugGfuuAfcaugAfaAfucccasusc-3′ (SEQ ID NO: 7), wherein a, c, g,and u are 2′-O-methyl (2′-OMe) A, C, G, or U; Af, Cf, Gf, and Uf are2′-fluoro A, C, G, or U; and s is a phosphorothioate linkage, therebyincreasing a 6-minute walk test (6MWT) in a subject suffering from aTTR-associated disease or at risk for developing a TTR-associateddisease.

The present invention is further illustrated by the following detaileddescription and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting the stability of the indicated RNAi agentsin a twenty-four hour tristosome stability assay.

FIG. 2A is a graph depicting the stability of the indicated RNAi agentsin a twenty-four hour rat cytosol stability assay and FIG. 2B is a graphdepicting the stability of the indicated RNAi agents in a twenty-fourhour tristosome stability assay.

FIG. 3 is a graph depicting TTR protein suppression in transgenic micethat express V30M variant of human TTR (V30M hTTR) followingadministration of a single subcutaneous dose of 1 mg/kg of the indicatedRNAi agents.

FIG. 4 is a graph depicting TTR protein suppression in transgenic micethat express hTTR V30M following administration of a single subcutaneousdose of 2.5 mg/kg of the indicated RNAi agents.

FIG. 5 is a graph depicting TTR protein suppression in transgenic micethat express hTTR V30M following administration of a weekly 2 mg/kg doseof AD-65492 for three weeks (QW×3).

FIG. 6A is graph depicting TTR protein suppression in transgenic micethat express hTTR V30M following subcutaneous administration of amonthly 0.3 mg/kg dose of the indicated RNAi agents for four months(QM×4 @ 0.3 mg/kg). FIG. 6B is graph depicting TTR protein suppressionin transgenic mice that express hTTR V30M following subcutaneousadministration of a monthly 1 mg/kg dose of the indicated RNAi agentsfor four months (QM×4 @ 1 mg/kg). FIG. 6C is graph depicting TTR proteinsuppression in transgenic mice that express hTTR V30M followingsubcutaneous administration of a monthly 3 mg/kg dose of the indicatedRNAi agents for four months (QM×4 @ 3 mg/kg).

FIG. 7 depicts the study design of AD-65492 and AD-66017 subcutaneousadministration to Cynomologous monkeys.

FIG. 8A is a graph depicting TTR protein suppression in Cynomologousmonkeys following administration of a single subcutaneous dose of 0.3mg/kg of the indicated RNAi agents. FIG. 8B is a graph depicting TTRprotein suppression in Cynomologous monkeys following administration ofa single subcutaneous dose of 1 mg/kg of AD-65492, a single subcutaneousdose of 1 mg/kg of AD-66017, or a single subcutaneous dose of 2.5 mg/kgof AD-51547. FIG. 8C is a graph depicting TTR protein suppression inCynomologous monkeys following administration of a single subcutaneousdose of 3 mg/kg of AD-65492, a single subcutaneous dose of 3 mg/kg ofAD-66017, or a single subcutaneous dose of 5 mg/kg of AD-51547.

FIG. 9A is a graph depicting TTR protein suppression in Cynomologousmonkeys following administration of a monthly subcutaneous dose of 1mg/kg for four months (QM×4) of AD-65492, a monthly subcutaneous dose of1 mg/kg for four months (QM×4) of AD-66017, or a daily dose of 5 mg/kgfor five days, followed by a weekly 5 mg/kg dose for four weeks (QD×5,QW×4) of AD-51547. FIG. 9B is a graph depicting TTR protein suppressionin Cynomologous monkeys following administration of a monthlysubcutaneous dose of 3 mg/kg for four months (QM×4) of the indicatedRNAi agents.

FIG. 10A is a graph depicting the maintenance of TTR suppression bysubcutaneous administration of a monthly 1 mg/kg dose of AD-65492 forfour months (QM×4; solid line) compared to TTR suppression after asingle 1 mg/kg subcutaneous dose of AD-65492 (dashed line) inCynomologous monkeys.

FIG. 10B is a graph depicting an additive effect of subcutaneousadministration of a monthly 1 mg/kg dose of AD-66017 for four months(QM×4; solid line) on TTR protein suppression compared to a singlesubcutaneous dose of 1 mg/kg of AD-66017 (dashed line) in Cynomologousmonkeys.

FIG. 11 is a graph depicting sustained serum TTR suppression inCynomologous monkeys following monthly subcutaneous administration of a1 mg/kg dose of AD-65492 for four months (QM×4), or monthly subcutaneousadministration of a 3 mg/kg dose of AD-65492 for four months (QM×4) ascompared to a single subcutaneously administered 1 mg/kg dose ofAD-65492 or a single subcutaneously administered 0.3 mg/kg dose ofAD-65492.

FIG. 12 depicts the study design of AD-65492 subcutaneous administrationto Cynomologous monkeys.

FIG. 13 is a graph depicting robust serum TTR suppression inCynomologous monkeys following monthly subcutaneous administration of a0.3 mg/kg dose of AD-65492 for six months (QM×6) or monthly subcutaneousadministration of a 0.6 mg/kg dose of AD-65492 for six months (QM×6) oradministration of a single 1 mg/kg initial dose of AD-65492 (QM×1)followed by a monthly 0.3 mg/kg dose of AD-65492 beginning at day 28post-initial dose for five months (QM×5).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides RNAi agents, e.g., double stranded RNAiagents, and compositions targeting the Transthyretin (TTR) gene. Thepresent invention also provides methods of inhibiting expression of TTRand methods of treating or preventing a TTR-associated disease in asubject using the RNAi agents, e.g., double stranded RNAi agents, of theinvention. The present invention is based, at least in part, on thediscovery that RNAi agents in which substantially all of the nucleotideson the sense strand and substantially all of the nucleotides of theantisense strand are modified nucleotides and that comprise no more than8 2′-fluoro modifications (e.g., no more than 7 2′-fluoro modifications,no more than 6 2′-fluoro modifications, no more than 5 2′-fluoromodifications, no more than 4 2′-fluoro modifications, no more than 52′-fluoro modifications, no more than 4 2′-fluoro modifications, no morethan 3 2′-fluoro modifications, or no more than 2 2′-fluoromodifications) on the sense strand, no more than 6 2′-fluoromodifications (e.g., no more than 5 2′-fluoro modifications, no morethan 4 2′-fluoro modifications, no more than 3 2′-fluoro modifications,or no more than 2 2′-fluoro modifications) on the antisense strand, twophosphorothioate linkages at the 5′-end of the sense strand, twophosphorothioate linkages at the 5′-end of the antisense strand, and aligand, e.g., a GalNAc₃ ligand, are shown herein to be effective inselectively silencing the activity of the TTR gene. These agents showsurprisingly enhanced TTR gene silencing activity. Without intending tobe limited by theory, it is believed that a combination orsub-combination of the foregoing modifications and the specific targetsites in these RNAi agents confer to the RNAi agents of the inventionimproved efficacy, stability, potency, and durability.

The following detailed description discloses how to make and usecompositions containing iRNAs to selectively inhibit the expression of aTTR gene, as well as compositions, uses, and methods for treatingsubjects having diseases and disorders that would benefit frominhibition and/or reduction of the expression of a TTR gene.

I. Definitions

In order that the present invention may be more readily understood,certain terms are first defined. In addition, it should be noted thatwhenever a value or range of values of a parameter are recited, it isintended that values and ranges intermediate to the recited values arealso intended to be part of this invention.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element, e.g., a plurality of elements.

The term “including” is used herein to mean, and is used interchangeablywith, the phrase “including but not limited to”.

The term “or” is used herein to mean, and is used interchangeably with,the term “and/or,” unless context clearly indicates otherwise.

The term “about” is used herein to mean within the typical ranges oftolerances in the art. For example, “about” can be understood as withinabout 2 standard deviations from the mean. In certain embodiments, aboutmeans+10%. In certain embodiments, about means+5%. When about is presentbefore a series of numbers or a range, it is understood that “about” canmodify each of the numbers in the series or range.

As used herein, a “transthyretin” (“TTR”) refers to the well known geneand protein. TTR is also known as prealbumin, HsT2651, PALB, and TBPA.TTR functions as a transporter of retinol-binding protein (RBP),thyroxine (T4) and retinol, and it also acts as a protease. The liversecretes TTR into the blood, and the choroid plexus secretes TTR intothe cerebrospinal fluid. TTR is also expressed in the pancreas and theretinal pigment epithelium. The greatest clinical relevance of TTR isthat both normal and mutant TTR protein can form amyloid fibrils thataggregate into extracellular deposits, causing amyloidosis. See, e.g.,Saraiva M. J. M. (2002) Expert Reviews in Molecular Medicine, 4(12):1-11for a review. The molecular cloning and nucleotide sequence of rattransthyretin, as well as the distribution of mRNA expression, wasdescribed by Dickson, P. W. et al. (1985) J. Biol. Chem.260(13)8214-8219. The X-ray crystal structure of human TTR was describedin Blake, C. C. et al. (1974) J Mol Biol 88, 1-12. The sequence of ahuman TTR mRNA transcript can be found at National Center forBiotechnology Information (NCBI) RefSeq accession number NM_000371(e.g., SEQ ID NOs:1 and 5). The sequence of mouse TTR mRNA can be foundat RefSeq accession number NM_013697.2, and the sequence of rat TTR mRNAcan be found at RefSeq accession number NM_012681.1. Additional examplesof TTR mRNA sequences are readily available using publicly availabledatabases, e.g., GenBank, UniProt, and OMIM.

As used herein, “target sequence” refers to a contiguous portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof a TTR gene, including mRNA that is a product of RNA processing of aprimary transcription product. In one embodiment, the target portion ofthe sequence will be at least long enough to serve as a substrate foriRNA-directed cleavage at or near that portion of the nucleotidesequence of an mRNA molecule formed during the transcription of a TTRgene. In one embodiment, the target sequence is within the proteincoding region of the TTR gene. In another embodiment, the targetsequence is within the 3′ UTR of the TTR gene.

The target sequence may be from about 9-36 nucleotides in length, e.g.,about 15-30 nucleotides in length. For example, the target sequence canbe from about 15-30 nucleotides, 15-29, 15-28, 15-27, 15-26, 15-25,15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29,18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30,19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20,20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21,21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22nucleotides in length. In some embodiments, the target sequence is about19 to about 30 nucleotides in length. In other embodiments, the targetsequence is about 19 to about 25 nucleotides in length. In still otherembodiments, the target sequence is about 19 to about 23 nucleotides inlength. In some embodiments, the target sequence is about 21 to about 23nucleotides in length. Ranges and lengths intermediate to the aboverecited ranges and lengths are also contemplated to be part of theinvention.

In some embodiments of the invention, the target sequence of a TTR genecomprises nucleotides 615-637 of SEQ ID NO:1 or nucleotides 505-527 ofSEQ ID NO:5 (i.e., 5′-GATGGGATTTCATGTAACCAAGA-3′; SEQ ID NO:4).

As used herein, the term “strand comprising a sequence” refers to anoligonucleotide comprising a chain of nucleotides that is described bythe sequence referred to using the standard nucleotide nomenclature.

“G,” “C,” “A,” “T” and “U” each generally stand for a nucleotide thatcontains guanine, cytosine, adenine, thymidine and uracil as a base,respectively. However, it will be understood that the term“ribonucleotide” or “nucleotide” can also refer to a modifiednucleotide, as further detailed below, or a surrogate replacement moiety(see, e.g., Table 2). The skilled person is well aware that guanine,cytosine, adenine, and uracil can be replaced by other moieties withoutsubstantially altering the base pairing properties of an oligonucleotidecomprising a nucleotide bearing such replacement moiety. For example,without limitation, a nucleotide comprising inosine as its base can basepair with nucleotides containing adenine, cytosine, or uracil. Hence,nucleotides containing uracil, guanine, or adenine can be replaced inthe nucleotide sequences of dsRNA featured in the invention by anucleotide containing, for example, inosine. In another example, adenineand cytosine anywhere in the oligonucleotide can be replaced withguanine and uracil, respectively to form G-U Wobble base pairing withthe target mRNA. Sequences containing such replacement moieties aresuitable for the compositions and methods featured in the invention.

The terms “iRNA,” “RNAi agent,” “iRNA agent,”, “RNA interference agent”as used interchangeably herein, refer to an agent that contains RNA asthat term is defined herein, and which mediates the targeted cleavage ofan RNA transcript via an RNA-induced silencing complex (RISC) pathway.iRNA directs the sequence-specific degradation of mRNA through a processknown as RNA interference (RNAi). The iRNA modulates, e.g., inhibits,the expression of a TTR gene in a cell, e.g., a cell within a subject,such as a mammalian subject.

In one embodiment, an RNAi agent of the invention includes a singlestranded RNA that interacts with a target RNA sequence, e.g., a TTRtarget mRNA sequence, to direct the cleavage of the target RNA. Withoutwishing to be bound by theory it is believed that long double strandedRNA introduced into cells is broken down into double stranded shortinterfering RNAs (siRNAs) comprising a sense strand and an antisensestrand by a Type III endonuclease known as Dicer (Sharp et al. (2001)Genes Dev. 15:485). Dicer, a ribonuclease-III-like enzyme, processesthese dsRNA into 19-23 base pair short interfering RNAs withcharacteristic two base 3′ overhangs (Bernstein, et al., (2001) Nature409:363). These siRNAs are then incorporated into an RNA-inducedsilencing complex (RISC) where one or more helicases unwind the siRNAduplex, enabling the complementary antisense strand to guide targetrecognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to theappropriate target mRNA, one or more endonucleases within the RISCcleave the target to induce silencing (Elbashir, et al., (2001) GenesDev. 15:188). Thus, in one aspect the invention relates to a singlestranded siRNA (ssRNA) (the antisense strand of an siRNA duplex)generated within a cell and which promotes the formation of a RISCcomplex to effect silencing of the target gene, i.e., a TTR gene.Accordingly, the term “siRNA” is also used herein to refer to an RNAi asdescribed above.

In another embodiment, the RNAi agent may be a single-stranded RNA thatis introduced into a cell or organism to inhibit a target mRNA.Single-stranded RNAi agents bind to the RISC endonuclease, Argonaute 2,which then cleaves the target mRNA. The single-stranded siRNAs aregenerally 15-30 nucleotides and are chemically modified. The design andtesting of single-stranded siRNAs are described in U.S. Pat. No.8,101,348 and in Lima et al., (2012) Cell 150:883-894, the entirecontents of each of which are hereby incorporated herein by reference.Any of the antisense nucleotide sequences described herein may be usedas a single-stranded RNA as described herein or as chemically modifiedby the methods described in Lima et al., (2012) Cell 150:883-894.

In another embodiment, an “iRNA” for use in the compositions, uses, andmethods of the invention is a double stranded RNA and is referred toherein as a “double stranded RNAi agent,” “double stranded RNA (dsRNA)molecule,” “dsRNA agent,” or “dsRNA”. The term “dsRNA” refers to acomplex of ribonucleic acid molecules, having a duplex structurecomprising two anti-parallel and substantially complementary nucleicacid strands, referred to as having “sense” and “antisense” orientationswith respect to a target RNA, i.e., a TTR gene. In some embodiments ofthe invention, a double stranded RNA (dsRNA) triggers the degradation ofa target RNA, e.g., an mRNA, through a post-transcriptionalgene-silencing mechanism referred to herein as RNA interference or RNAi.

In general, the majority of nucleotides of each strand of a dsRNAmolecule are ribonucleotides, but as described in detail herein, each orboth strands can also include one or more non-ribonucleotides, e.g., adeoxyribonucleotide and/or a modified nucleotide. In addition, as usedin this specification, an “RNAi agent” may include ribonucleotides withchemical modifications; an RNAi agent may include substantialmodifications at multiple nucleotides.

As used herein, the term “modified nucleotide” refers to a nucleotidehaving, independently, a modified sugar moiety, a modifiedinternucleotide linkage, and/or a modified nucleobase. Thus, the termmodified nucleotide encompasses substitutions, additions or removal of,e.g., a functional group or atom, to internucleoside linkages, sugarmoieties, or nucleobases. The modifications suitable for use in theagents of the invention include all types of modifications disclosedherein or known in the art. Any such modifications, as used in a siRNAtype molecule, are encompassed by “RNAi agent” for the purposes of thisspecification and claims.

The duplex region may be of any length that permits specific degradationof a desired target RNA through a RISC pathway, and may range from about9 to 36 base pairs in length, e.g., about 15-30 base pairs in length,for example, about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairsin length, such as about 15-30, 15-29, 15-28, 15-27, 15-26, 15-25,15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29,18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30,19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20,20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21,21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 basepairs in length. Ranges and lengths intermediate to the above recitedranges and lengths are also contemplated to be part of the invention.

The two strands forming the duplex structure may be different portionsof one larger RNA molecule, or they may be separate RNA molecules. Wherethe two strands are part of one larger molecule, and therefore areconnected by an uninterrupted chain of nucleotides between the 3′-end ofone strand and the 5′-end of the respective other strand forming theduplex structure, the connecting RNA chain is referred to as a “hairpinloop.” A hairpin loop can comprise at least one unpaired nucleotide. Insome embodiments, the hairpin loop can comprise at least 2, at least 3,at least 4, at least 5, at least 6, at least 7, at least 8, at least 9,at least 10, at least 20, at least 23 or more unpaired nucleotides. Insome embodiments, the hairpin loop can be 10 or fewer nucleotides. Insome embodiments, the hairpin loop can be 8 or fewer unpairednucleotides. In some embodiments, the hairpin loop can be 4-10 unpairednucleotides. In some embodiments, the hairpin loop can be 4-8nucleotides.

Where the two substantially complementary strands of a dsRNA arecomprised by separate RNA molecules, those molecules need not, but canbe covalently connected. Where the two strands are connected covalentlyby means other than an uninterrupted chain of nucleotides between the3′-end of one strand and the 5′-end of the respective other strandforming the duplex structure, the connecting structure is referred to asa “linker.” The RNA strands may have the same or a different number ofnucleotides. The maximum number of base pairs is the number ofnucleotides in the shortest strand of the dsRNA minus any overhangs thatare present in the duplex. In addition to the duplex structure, an RNAimay comprise one or more nucleotide overhangs.

In one embodiment, an RNAi agent of the invention is a dsRNA, eachstrand of which is 24-30 nucleotides in length, that interacts with atarget RNA sequence, e.g., a TTR target mRNA sequence, to direct thecleavage of the target RNA. Without wishing to be bound by theory, longdouble stranded RNA introduced into cells is broken down into siRNA by aType III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev.15:485). Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into19-23 base pair short interfering RNAs with characteristic two base 3′overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs arethen incorporated into an RNA-induced silencing complex (RISC) where oneor more helicases unwind the siRNA duplex, enabling the complementaryantisense strand to guide target recognition (Nykanen, et al., (2001)Cell 107:309). Upon binding to the appropriate target mRNA, one or moreendonucleases within the RISC cleave the target to induce silencing(Elbashir, et al., (2001) Genes Dev. 15:188). In one embodiment of theRNAi agent, at least one strand comprises a 3′ overhang of at least 1nucleotide. In another embodiment, at least one strand comprises a 3′overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11,12, 13, 14, or 15 nucleotides. In other embodiments, at least one strandof the RNAi agent comprises a 5′ overhang of at least 1 nucleotide. Incertain embodiments, at least one strand comprises a 5′ overhang of atleast 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or15 nucleotides. In still other embodiments, both the 3′ and the 5′ endof one strand of the RNAi agent comprise an overhang of at least 1nucleotide.

In one embodiment, an RNAi agent of the invention is a dsRNA agent, eachstrand of which comprises 19-23 nucleotides that interacts with aTTR RNAsequence to direct the cleavage of the target RNA. Without wishing to bebound by theory, long double stranded RNA introduced into cells isbroken down into siRNA by a Type III endonuclease known as Dicer (Sharpet al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease-III-like enzyme,processes the dsRNA into 19-23 base pair short interfering RNAs withcharacteristic two base 3′ overhangs (Bernstein, et al., (2001) Nature409:363). The siRNAs are then incorporated into an RNA-induced silencingcomplex (RISC) where one or more helicases unwind the siRNA duplex,enabling the complementary antisense strand to guide target recognition(Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriatetarget mRNA, one or more endonucleases within the RISC cleave the targetto induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). In oneembodiment, an RNAi agent of the invention is a dsRNA of 24-30nucleotides that interacts with a TTR RNA sequence to direct thecleavage of the target RNA.

As used herein, the term “nucleotide overhang” refers to at least oneunpaired nucleotide that protrudes from the duplex structure of an iRNA,e.g., a dsRNA. For example, when a 3′-end of one strand of a dsRNAextends beyond the 5′-end of the other strand, or vice versa, there is anucleotide overhang. A dsRNA can comprise an overhang of at least onenucleotide; alternatively the overhang can comprise at least twonucleotides, at least three nucleotides, at least four nucleotides, atleast five nucleotides or more. A nucleotide overhang can comprise orconsist of a nucleotide/nucleoside analog, including adeoxynucleotide/nucleoside. The overhang(s) can be on the sense strand,the antisense strand or any combination thereof. Furthermore, thenucleotide(s) of an overhang can be present on the 5′-end, 3′-end orboth ends of either an antisense or sense strand of a dsRNA. In oneembodiment of the dsRNA, at least one strand comprises a 3′ overhang ofat least 1 nucleotide. In another embodiment, at least one strandcomprises a 3′ overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6,7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, atleast one strand of the RNAi agent comprises a 5′ overhang of at least 1nucleotide. In certain embodiments, at least one strand comprises a 5′overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11,12, 13, 14, or 15 nucleotides. In still other embodiments, both the 3′and the 5′ end of one strand of the RNAi agent comprise an overhang ofat least 1 nucleotide.

In one embodiment, the antisense strand of a dsRNA has a 1-10nucleotide, e.g., 0-3, 1-3, 2-4, 2-5, 4-10, 5-10, e.g., a 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 nucleotide, overhang at the 3′-end and/or the 5′-end.In one embodiment, the sense strand of a dsRNA has a 1-10 nucleotide,e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide, overhang at the3′-end and/or the 5′-end. In another embodiment, one or more of thenucleotides in the overhang is replaced with a nucleoside thiophosphate.

In certain embodiments, the overhang on the sense strand or theantisense strand, or both, can include extended lengths longer than 10nucleotides, e.g., 1-30 nucleotides, 2-30 nucleotides, 10-30nucleotides, or 10-15 nucleotides in length. In certain embodiments, anextended overhang is on the sense strand of the duplex. In certainembodiments, an extended overhang is present on the 3′ end of the sensestrand of the duplex. In certain embodiments, an extended overhang ispresent on the 5′ end of the sense strand of the duplex. In certainembodiments, an extended overhang is on the antisense strand of theduplex. In certain embodiments, an extended overhang is present on the3′ end of the antisense strand of the duplex. In certain embodiments, anextended overhang is present on the 5′ end of the antisense strand ofthe duplex. In certain embodiments, one or more of the nucleotides inthe overhang is replaced with a nucleoside thiophosphate. In certainembodiments, the overhang includes a self-complementary portion suchthat the overhang is capable of forming a hairpin structure that isstable under physiological conditions.

“Blunt” or “blunt end” means that there are no unpaired nucleotides atthat end of the double stranded RNAi agent, i.e., no nucleotideoverhang. A “blunt ended” RNAi agent is a dsRNA that is double strandedover its entire length, i.e., no nucleotide overhang at either end ofthe molecule. The RNAi agents of the invention include RNAi agents withnucleotide overhangs at one end (i.e., agents with one overhang and oneblunt end) or with nucleotide overhangs at both ends.

The term “antisense strand” or “guide strand” refers to the strand of aniRNA, e.g., a dsRNA, which includes a region that is substantiallycomplementary to a target sequence, e.g., a TTR mRNA. As used herein,the term “region of complementarity” refers to the region on theantisense strand that is substantially complementary to a sequence, forexample a target sequence, e.g., a TTR nucleotide sequence, as definedherein. Where the region of complementarity is not fully complementaryto the target sequence, the mismatches can be in the internal orterminal regions of the molecule. Generally, the most toleratedmismatches are in the terminal regions, e.g., within 5, 4, 3, 2, or 1nucleotides of the 5′- and/or 3′-terminus of the iRNA. In oneembodiment, a double stranded RNAi agent of the invention include anucleotide mismatch in the antisense strand. In another embodiment, adouble stranded RNAi agent of the invention include a nucleotidemismatch in the sense strand. In one embodiment, the nucleotide mismatchis, for example, within 5, 4, 3, 2, or 1 nucleotides from the3′-terminus of the iRNA. In another embodiment, the nucleotide mismatchis, for example, in the 3′-terminal nucleotide of the iRNA.

The term “sense strand,” or “passenger strand” as used herein, refers tothe strand of an iRNA that includes a region that is substantiallycomplementary to a region of the antisense strand as that term isdefined herein.

As used herein, the term “cleavage region” refers to a region that islocated immediately adjacent to the cleavage site. The cleavage site isthe site on the target at which cleavage occurs. In some embodiments,the cleavage region comprises three bases on either end of, andimmediately adjacent to, the cleavage site. In some embodiments, thecleavage region comprises two bases on either end of, and immediatelyadjacent to, the cleavage site. In some embodiments, the cleavage sitespecifically occurs at the site bound by nucleotides 10 and 11 of theantisense strand, and the cleavage region comprises nucleotides 11, 12and 13.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence inrelation to a second nucleotide sequence, refers to the ability of anoligonucleotide or polynucleotide comprising the first nucleotidesequence to hybridize and form a duplex structure under certainconditions with an oligonucleotide or polynucleotide comprising thesecond nucleotide sequence, as will be understood by the skilled person.Such conditions can, for example, be stringent conditions, wherestringent conditions can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. for 12-16 hours followed by washing (see, e.g.,“Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) ColdSpring Harbor Laboratory Press). Other conditions, such asphysiologically relevant conditions as can be encountered inside anorganism, can apply. The skilled person will be able to determine theset of conditions most appropriate for a test of complementarity of twosequences in accordance with the ultimate application of the hybridizednucleotides.

Complementary sequences within an iRNA, e.g., within a dsRNA asdescribed herein, include base-pairing of the oligonucleotide orpolynucleotide comprising a first nucleotide sequence to anoligonucleotide or polynucleotide comprising a second nucleotidesequence over the entire length of one or both nucleotide sequences.Such sequences can be referred to as “fully complementary” with respectto each other herein. However, where a first sequence is referred to as“substantially complementary” with respect to a second sequence herein,the two sequences can be fully complementary, or they can form one ormore, but generally not more than 5, 4, 3 or 2 mismatched base pairsupon hybridization for a duplex up to 30 base pairs, while retaining theability to hybridize under the conditions most relevant to theirultimate application, e.g., inhibition of gene expression via a RISCpathway. However, where two oligonucleotides are designed to form, uponhybridization, one or more single stranded overhangs, such overhangsshall not be regarded as mismatches with regard to the determination ofcomplementarity. For example, a dsRNA comprising one oligonucleotide 21nucleotides in length and another oligonucleotide 23 nucleotides inlength, wherein the longer oligonucleotide comprises a sequence of 21nucleotides that is fully complementary to the shorter oligonucleotide,can yet be referred to as “fully complementary” for the purposesdescribed herein.

“Complementary” sequences, as used herein, can also include, or beformed entirely from, non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in so far as the aboverequirements with respect to their ability to hybridize are fulfilled.Such non-Watson-Crick base pairs include, but are not limited to, G:UWobble or Hoogstein base pairing.

The terms “complementary,” “fully complementary” and “substantiallycomplementary” herein can be used with respect to the base matchingbetween the sense strand and the antisense strand of a dsRNA, or betweenthe antisense strand of an iRNA agent and a target sequence, as will beunderstood from the context of their use.

As used herein, a polynucleotide that is “substantially complementary toat least part of” a messenger RNA (mRNA) refers to a polynucleotide thatis substantially complementary to a contiguous portion of the mRNA ofinterest (e.g., an mRNA encoding a TTR gene). For example, apolynucleotide is complementary to at least a part of a TTR mRNA if thesequence is substantially complementary to a non-interrupted portion ofan mRNA encoding a TTR gene.

Accordingly, in some embodiments, the antisense polynucleotidesdisclosed herein are fully complementary to the target TTR sequence. Inother embodiments, the antisense polynucleotides disclosed herein arefully complementary to SEQ ID NO:2 (5′-UGGGAUUUCAUGUAACCAAGA-3′). In oneembodiment, the antisense polynucleotide sequence is5′-UCUUGGUUACAUGAAAUCCCAUC-3′ (SEQ ID NO:3).

In other embodiments, the the antisense polynucleotides disclosed hereinare substantially complementary to the target TTR sequence and comprisea contiguous nucleotide sequence which is at least about 80%complementary over its entire length to the equivalent region of thenucleotide sequence of any one of SEQ ID NO:2(5′-UGGGAUUUCAUGUAACCAAGA-3′), or a fragment of any one of SEQ ID NOs:1,2, and 5, such as about 85%, about 86%, about 87%, about 88%, about 89%,about 90%, about % 91%, about 92%, about 93%, about 94%, about 95%,about 96%, about 97%, about 98%, or about 99% complementary.

In one embodiment, an RNAi agent of the invention includes a sensestrand that is substantially complementary to an antisensepolynucleotide which, in turn, is complementary to a target TTRsequence, and wherein the sense strand polynucleotide comprises acontiguous nucleotide sequence which is at least about 80% complementaryover its entire length to the equivalent region of the nucleotidesequence of any one of the sequences in Tables 1, 3, 5, 6, and 7, suchas about 85%, about 86%, about 87%, about 88%, about 89%, about 90%,about % 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98%, or about 99% complementary.

In another embodiment, an RNAi agent of the invention includes anantisense strand that is substantially complementary to the target TTRsequence and comprise a contiguous nucleotide sequence which is at leastabout 80% complementary over its entire length to the equivalent regionof the nucleotide sequence of any one of the sequences in Table 1 and 3,such as about 85%, about 86%, about 87%, about 88%, about 89%, about90%, about % 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98%, or about 99% complementary.

In some embodiments, the majority of nucleotides of each strand areribonucleotides, but as described in detail herein, each or both strandscan also include one or more non-ribonucleotides, e.g., adeoxyribonucleotide and/or a modified nucleotide. In addition, an “iRNA”may include ribonucleotides with chemical modifications. Suchmodifications may include all types of modifications disclosed herein orknown in the art. Any such modifications, as used in an iRNA molecule,are encompassed by “iRNA” for the purposes of this specification andclaims.

In one aspect of the invention, an agent for use in the methods andcompositions of the invention is a single-stranded antisense nucleicacid molecule that inhibits a target mRNA via an antisense inhibitionmechanism. The single-stranded antisense RNA molecule is complementaryto a sequence within the target mRNA. The single-stranded antisenseoligonucleotides can inhibit translation in a stoichiometric manner bybase pairing to the mRNA and physically obstructing the translationmachinery, see Dias, N. et al., (2002) Mol Cancer Ther 1:347-355. Thesingle-stranded antisense RNA molecule may be about 15 to about 30nucleotides in length and have a sequence that is complementary to atarget sequence. For example, the single-stranded antisense RNA moleculemay comprise a sequence that is at least about 15, 16, 17, 18, 19, 20,or more contiguous nucleotides from any one of the antisense sequencesdescribed herein.

A “TTR-associated disease,” as used herein, is intended to include anydisease associated with the TTR gene or protein. Such a disease may becaused, for example, by excess production of the TTR protein, by TTRgene mutations, by abnormal cleavage of the TTR protein, by abnormalinteractions between TTR and other proteins or other endogenous orexogenous substances. A “TTR-associated disease” includes any type ofTTR amyloidosis (ATTR) wherein TTR plays a role in the formation ofabnormal extracellular aggregates or amyloid deposits. TTR-associateddiseases include senile systemic amyloidosis (SSA), systemic familialamyloidosis, familial amyloidotic polyneuropathy (FAP), familialamyloidotic cardiomyopathy (FAC), leptomeningeal/Central Nervous System(CNS) amyloidosis, amyloidotic vitreous opacities, carpal tunnelsyndrome, and hyperthyroxinemia. Symptoms of TTR amyloidosis includesensory neuropathy (e.g., paresthesia, hypesthesia in distal limbs),autonomic neuropathy (e.g., gastrointestinal dysfunction, such asgastric ulcer, or orthostatic hypotension), motor neuropathy, seizures,dementia, myelopathy, polyneuropathy, carpal tunnel syndrome, autonomicinsufficiency, cardiomyopathy, vitreous opacities, renal insufficiency,nephropathy, substantially reduced mBMI (modified Body Mass Index),cranial nerve dysfunction, and corneal lattice dystrophy.

II. iRNAs of the Invention

The present invention provides iRNAs which selectively inhibit theexpression of one or more TTR genes. In one embodiment, the iRNA agentincludes double stranded ribonucleic acid (dsRNA) molecules forinhibiting the expression of a TTR gene in a cell, such as a cell withina subject, e.g., a mammal, such as a human having a TTR-associateddisease. The dsRNA includes an antisense strand having a region ofcomplementarity which is complementary to at least a part of an mRNAformed in the expression of a TTR gene. The region of complementarity isabout 30 nucleotides or less in length (e.g., about 30, 29, 28, 27, 26,25, 24, 23, 22, 21, 20, 19, or 18 nucleotides or less in length). Uponcontact with a cell expressing the TTR gene, the iRNA selectivelyinhibits the expression of the TTR gene (e.g., a human, a primate, anon-primate, or a bird TTR gene) by at least about 10% as assayed by,for example, a PCR or branched DNA (bDNA)-based method, or by aprotein-based method, such as by immunofluorescence analysis, using, forexample, Western Blotting or flowcytometric techniques.

A dsRNA includes two RNA strands that are complementary and hybridize toform a duplex structure under conditions in which the dsRNA will beused. One strand of a dsRNA (the antisense strand) includes a region ofcomplementarity that is substantially complementary, and generally fullycomplementary, to a target sequence. The target sequence can be derivedfrom the sequence of an mRNA formed during the expression of a TTR gene.The other strand (the sense strand) includes a region that iscomplementary to the antisense strand, such that the two strandshybridize and form a duplex structure when combined under suitableconditions. As described elsewhere herein and as known in the art, thecomplementary sequences of a dsRNA can also be contained asself-complementary regions of a single nucleic acid molecule, as opposedto being on separate oligonucleotides.

Generally, the duplex structure is between 15 and 30 base pairs inlength, e.g., between, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23,15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27,18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28,19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29,20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29,21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length.Ranges and lengths intermediate to the above recited ranges and lengthsare also contemplated to be part of the invention.

Similarly, the region of complementarity to the target sequence isbetween 15 and 30 nucleotides in length, e.g., between 15-29, 15-28,15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18,15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22,18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23,19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24,21-23, or 21-22 nucleotides in length. Ranges and lengths intermediateto the above recited ranges and lengths are also contemplated to be partof the invention.

In some embodiments, the dsRNA is about 15 to about 20 nucleotides inlength, or about 25 to about 30 nucleotides in length. In general, thedsRNA is long enough to serve as a substrate for the Dicer enzyme. Forexample, it is well-known in the art that dsRNAs longer than about 21-23nucleotides in length may serve as substrates for Dicer. As theordinarily skilled person will also recognize, the region of an RNAtargeted for cleavage will most often be part of a larger RNA molecule,often an mRNA molecule. Where relevant, a “part” of an mRNA target is acontiguous sequence of an mRNA target of sufficient length to allow itto be a substrate for RNAi-directed cleavage (i.e., cleavage through aRISC pathway).

One of skill in the art will also recognize that the duplex region is aprimary functional portion of a dsRNA, e.g., a duplex region of about 9to 36 base pairs, e.g., about 10-36, 11-36, 12-36, 13-36, 14-36, 15-36,9-35, 10-35, 11-35, 12-35, 13-35, 14-35, 15-35, 9-34, 10-34, 11-34,12-34, 13-34, 14-34, 15-34, 9-33, 10-33, 11-33, 12-33, 13-33, 14-33,15-33, 9-32, 10-32, 11-32, 12-32, 13-32, 14-32, 15-32, 9-31, 10-31,11-31, 12-31, 13-32, 14-31, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26,15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30,18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20,19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21,19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22,20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22base pairs. Thus, in one embodiment, to the extent that it becomesprocessed to a functional duplex, of e.g., 15-30 base pairs, thattargets a desired RNA for cleavage, an RNA molecule or complex of RNAmolecules having a duplex region greater than 30 base pairs is a dsRNA.Thus, an ordinarily skilled artisan will recognize that in oneembodiment, a miRNA is a dsRNA. In another embodiment, a dsRNA is not anaturally occurring miRNA. In another embodiment, an iRNA agent usefulto target TTR gene expression is not generated in the target cell bycleavage of a larger dsRNA.

A dsRNA as described herein can further include one or moresingle-stranded nucleotide overhangs e.g., 1, 2, 3, or 4 nucleotides.dsRNAs having at least one nucleotide overhang can have unexpectedlysuperior inhibitory properties relative to their blunt-endedcounterparts. A nucleotide overhang can comprise or consist of anucleotide/nucleoside analog, including a deoxynucleotide/nucleoside.The overhang(s) can be on the sense strand, the antisense strand or anycombination thereof. Furthermore, the nucleotide(s) of an overhang canbe present on the 5′-end, 3′-end or both ends of either an antisense orsense strand of a dsRNA. In certain embodiments, longer, extendedoverhangs are possible.

A dsRNA can be synthesized by standard methods known in the art asfurther discussed below, e.g., by use of an automated DNA synthesizer,such as are commercially available from, for example, Biosearch, AppliedBiosystems, Inc.

iRNA compounds of the invention may be prepared using a two-stepprocedure. First, the individual strands of the double stranded RNAmolecule are prepared separately. Then, the component strands areannealed. The individual strands of the siRNA compound can be preparedusing solution-phase or solid-phase organic synthesis or both. Organicsynthesis offers the advantage that the oligonucleotide strandscomprising unnatural or modified nucleotides can be easily prepared.Single-stranded oligonucleotides of the invention can be prepared usingsolution-phase or solid-phase organic synthesis or both.

In one aspect, a dsRNA of the invention includes at least two nucleotidesequences, a sense sequence and an anti-sense sequence. The sense strandis selected from the group of sequences provided in any one of Tables 1,3, 5, 6, and 7, and the corresponding antisense strand of the sensestrand is selected from the group of sequences of any one of Tables 1,3, 5, 6, and 7. In this aspect, one of the two sequences iscomplementary to the other of the two sequences, with one of thesequences being substantially complementary to a sequence of an mRNAgenerated in the expression of a TTR gene. As such, in this aspect, adsRNA will include two oligonucleotides, where one oligonucleotide isdescribed as the sense strand in any one of Tables 1, 3, 5, 6, and 7,and the second oligonucleotide is described as the correspondingantisense strand of the sense strand in any one of Tables 1, 3, 5, 6,and 7. In one embodiment, the substantially complementary sequences ofthe dsRNA are contained on separate oligonucleotides. In anotherembodiment, the substantially complementary sequences of the dsRNA arecontained on a single oligonucleotide.

It will be understood that, although some of the sequences in Tables 1,3, 5, 6, and 7 are described as modified and/or conjugated sequences,the RNA of the iRNA of the invention e.g., a dsRNA of the invention, maycomprise any one of the sequences set forth in Tables 1, 3, 5, 6, and 7that is un-modified, un-conjugated, and/or modified and/or conjugateddifferently than described therein.

The skilled person is well aware that dsRNAs having a duplex structureof between about 20 and 23 base pairs, e.g., 21, base pairs have beenhailed as particularly effective in inducing RNA interference (Elbashiret al., EMBO 2001, 20:6877-6888). However, others have found thatshorter or longer RNA duplex structures can also be effective (Chu andRana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226).In the embodiments described above, by virtue of the nature of theoligonucleotide sequences provided in any one of Tables 1, 3, 5, 6, and7, dsRNAs described herein can include at least one strand of a lengthof minimally 21 nucleotides. It can be reasonably expected that shorterduplexes having one of the sequences of any one of Tables 1, 3, 5, 6,and 7 minus only a few nucleotides on one or both ends can be similarlyeffective as compared to the dsRNAs described above. Hence, dsRNAshaving a sequence of at least 15, 16, 17, 18, 19, 20, or more contiguousnucleotides derived from one of the sequences of any one of Tables 1, 3,5, 6, and 7, and differing in their ability to inhibit the expression ofa TTR gene by not more than about 5, 10, 15, 20, 25, or 30% inhibitionfrom a dsRNA comprising the full sequence, are contemplated to be withinthe scope of the present invention.

In addition, the RNAs provided in any one of Tables 1, 3, 5, 6, and 7identify a site(s) in a TTR transcript that is susceptible toRISC-mediated cleavage. As such, the present invention further featuresiRNAs that target within one of these sites. As used herein, an iRNA issaid to target within a particular site of an RNA transcript if the iRNApromotes cleavage of the transcript anywhere within that particularsite. Such an iRNA will generally include at least about 15 contiguousnucleotides from one of the sequences provided in any one of Tables 1,3, 5, 6, and 7 coupled to additional nucleotide sequences taken from theregion contiguous to the selected sequence in a TTR gene.

While a target sequence is generally about 15-30 nucleotides in length,there is wide variation in the suitability of particular sequences inthis range for directing cleavage of any given target RNA. Varioussoftware packages and the guidelines set out herein provide guidance forthe identification of optimal target sequences for any given genetarget, but an empirical approach can also be taken in which a “window”or “mask” of a given size (as a non-limiting example, 21 nucleotides) isliterally or figuratively (including, e.g., in silico) placed on thetarget RNA sequence to identify sequences in the size range that canserve as target sequences. By moving the sequence “window” progressivelyone nucleotide upstream or downstream of an initial target sequencelocation, the next potential target sequence can be identified, untilthe complete set of possible sequences is identified for any giventarget size selected. This process, coupled with systematic synthesisand testing of the identified sequences (using assays as describedherein or as known in the art) to identify those sequences that performoptimally can identify those RNA sequences that, when targeted with aniRNA agent, mediate the best inhibition of target gene expression. Thus,while the sequences identified, for example, in any one of Tables 1, 3,5, 6, and 7 represent effective target sequences, it is contemplatedthat further optimization of inhibition efficiency can be achieved byprogressively “walking the window” one nucleotide upstream or downstreamof the given sequences to identify sequences with equal or betterinhibition characteristics.

Further, it is contemplated that for any sequence identified, e.g., inany one of Tables 1, 3, 5, 6, and 7, further optimization could beachieved by systematically either adding or removing nucleotides togenerate longer or shorter sequences and testing those sequencesgenerated by walking a window of the longer or shorter size up or downthe target RNA from that point. Again, coupling this approach togenerating new candidate targets with testing for effectiveness of iRNAsbased on those target sequences in an inhibition assay as known in theart and/or as described herein can lead to further improvements in theefficiency of inhibition. Further still, such optimized sequences can beadjusted by, e.g., the introduction of modified nucleotides as describedherein or as known in the art, addition or changes in overhang, or othermodifications as known in the art and/or discussed herein to furtheroptimize the molecule (e.g., increasing serum stability or circulatinghalf-life, increasing thermal stability, enhancing transmembranedelivery, targeting to a particular location or cell type, increasinginteraction with silencing pathway enzymes, increasing release fromendosomes) as an expression inhibitor.

An iRNA as described herein can contain one or more mismatches to thetarget sequence. In one embodiment, an iRNA as described herein containsno more than 3 mismatches. If the antisense strand of the iRNA containsmismatches to a target sequence, it is preferable that the area ofmismatch is not located in the center of the region of complementarity.If the antisense strand of the iRNA contains mismatches to the targetsequence, it is preferable that the mismatch be restricted to be withinthe last 5 nucleotides from either the 5′- or 3′-end of the region ofcomplementarity. For example, for a 23 nucleotide iRNA agent the strandwhich is complementary to a region of a TTR gene, generally does notcontain any mismatch within the central 13 nucleotides. The methodsdescribed herein or methods known in the art can be used to determinewhether an iRNA containing a mismatch to a target sequence is effectivein inhibiting the expression of a TTR gene. Consideration of theefficacy of iRNAs with mismatches in inhibiting expression of a TTR geneis important, especially if the particular region of complementarity ina TTR gene is known to have polymorphic sequence variation within thepopulation.

III. Modified iRNAs of the Invention

In one embodiment, the RNA of the iRNA of the invention e.g., a dsRNA,is un-modified, and does not comprise, e.g., chemical modificationsand/or conjugations known in the art and described herein. In anotherembodiment, the RNA of an iRNA of the invention, e.g., a dsRNA, ischemically modified to enhance stability or other beneficialcharacteristics. In certain embodiments of the invention, substantiallyall of the nucleotides of an iRNA of the invention are modified. Inother embodiments of the invention, all of the nucleotides of an iRNA ofthe invention are modified. In some embodiments, substantially all ofthe nucleotides of an iRNA of the invention are modified and the iRNAcomprises no more than 8 2′-fluoro modifications (e.g., no more than 72′-fluoro modifications, no more than 6 2′-fluoro modifications, no morethan 5 2′-fluoro modification, no more than 4 2′-fluoro modifications,no more than 3 2′-fluoro modifications, or no more than 2 2′-fluoromodifications) on the sense strand and no more than 6 2′-fluoromodifications (e.g., no more than 5 2′-fluoro modifications, no morethan 4 2′-fluoro modifications, no more than 3 2′-fluoro modifications,or no more than 2 2′-fluoro modifications) on the antisense strand. Inother embodiments, all of the nucleotides of an iRNA of the inventionare modified and the iRNA comprises no more than 8 2′-fluoromodifications (e.g., no more than 7 2′-fluoro modifications, no morethan 6 2′-fluoro modifications, no more than 5 2′-fluoro modification,no more than 4 2′-fluoro modifications, no more than 3 2′-fluoromodifications, or no more than 2 2′-fluoro modifications) on the sensestrand and no more than 6 2′-fluoro modifications (e.g., no more than 52′-fluoro modifications, no more than 4 2′-fluoro modifications, no morethan 3 2′-fluoro modifications, or no more than 2 2′-fluoromodifications) on the antisense strand. iRNAs of the invention in which“substantially all of the nucleotides are modified” are largely but notwholly modified and can include not more than 5, 4, 3, 2, or 1unmodified nucleotides.

The nucleic acids featured in the invention can be synthesized and/ormodified by methods well established in the art, such as those describedin “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al.(Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is herebyincorporated herein by reference. Modifications include, for example,end modifications, e.g., 5′-end modifications (phosphorylation,conjugation, inverted linkages) or 3′-end modifications (conjugation,DNA nucleotides, inverted linkages, etc.); base modifications, e.g.,replacement with stabilizing bases, destabilizing bases, or bases thatbase pair with an expanded repertoire of partners, removal of bases(abasic nucleotides), or conjugated bases; sugar modifications (e.g., atthe 2′-position or 4′-position) or replacement of the sugar; and/orbackbone modifications, including modification or replacement of thephosphodiester linkages. Specific examples of iRNA compounds useful inthe embodiments described herein include, but are not limited to RNAscontaining modified backbones or no natural internucleoside linkages.RNAs having modified backbones include, among others, those that do nothave a phosphorus atom in the backbone. For the purposes of thisspecification, and as sometimes referenced in the art, modified RNAsthat do not have a phosphorus atom in their internucleoside backbone canalso be considered to be oligonucleosides. In some embodiments, amodified iRNA will have a phosphorus atom in its internucleosidebackbone.

Modified RNA backbones include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′-linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170;6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423;6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294;6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat. RE39,464, the entire contents of each of which are hereby incorporatedherein by reference.

Modified RNA backbones that do not include a phosphorus atom thereinhave backbones that are formed by short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatoms and alkyl or cycloalkylinternucleoside linkages, or one or more short chain heteroatomic orheterocyclic internucleoside linkages. These include those havingmorpholino linkages (formed in part from the sugar portion of anucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

Representative U.S. patents that teach the preparation of the aboveoligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and,5,677,439, the entire contents of each of which are hereby incorporatedherein by reference.

In other embodiments, suitable RNA mimetics are contemplated for use iniRNAs, in which both the sugar and the internucleoside linkage, i.e.,the backbone, of the nucleotide units are replaced with novel groups.The base units are maintained for hybridization with an appropriatenucleic acid target compound. One such oligomeric compound, an RNAmimetic that has been shown to have excellent hybridization properties,is referred to as a peptide nucleic acid (PNA). In PNA compounds, thesugar backbone of an RNA is replaced with an amide containing backbone,in particular an aminoethylglycine backbone. The nucleobases areretained and are bound directly or indirectly to aza nitrogen atoms ofthe amide portion of the backbone. Representative U.S. patents thatteach the preparation of PNA compounds include, but are not limited to,U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, the entire contentsof each of which are hereby incorporated herein by reference. AdditionalPNA compounds suitable for use in the iRNAs of the invention aredescribed in, for example, in Nielsen et al., Science, 1991, 254,1497-1500.

Some embodiments featured in the invention include RNAs withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂— [known asa methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of theabove-referenced U.S. Pat. No. 5,489,677, and the amide backbones of theabove-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAsfeatured herein have morpholino backbone structures of theabove-referenced U.S. Pat. No. 5,034,506.

Modified RNAs can also contain one or more substituted sugar moieties.The iRNAs, e.g., dsRNAs, featured herein can include one of thefollowing at the 2′-position: OH; F; O-, S-, or N-alkyl; O-, S-, orN-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl can be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Exemplary suitable modificationsinclude O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)._(n)OCH₃, O(CH₂)_(n)NH₂,O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where nand m are from 1 to about 10. In other embodiments, dsRNAs include oneof the following at the 2′ position: C₁ to C₁₀ lower alkyl, substitutedlower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN,Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of aniRNA, or a group for improving the pharmacodynamic properties of aniRNA, and other substituents having similar properties. In someembodiments, the modification includes a 2′-methoxyethoxy(2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martinet al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxygroup. Another exemplary modification is 2′-dimethylaminooxyethoxy,i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described inexamples herein below, and 2′-dimethylaminoethoxyethoxy (also known inthe art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂.

Other modifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications can alsobe made at other positions on the RNA of an iRNA, particularly the 3′position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linkeddsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs can alsohave sugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. Representative U.S. patents that teach thepreparation of such modified sugar structures include, but are notlimited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which arecommonly owned with the instant application. The entire contents of eachof the foregoing are hereby incorporated herein by reference.

The RNA of an iRNA of the invention can also include nucleobase (oftenreferred to in the art simply as “base”) modifications or substitutions.As used herein, “unmodified” or “natural” nucleobases include the purinebases adenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U). Modified nucleobases include othersynthetic and natural nucleobases such as deoxy-thymine (dT),5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives ofadenine and guanine, 2-propyl and other alkyl derivatives of adenine andguanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouraciland cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine andthymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines andguanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Furthernucleobases include those disclosed in U.S. Pat. No. 3,687,808, thosedisclosed in Modified Nucleosides in Biochemistry, Biotechnology andMedicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in TheConcise Encyclopedia Of Polymer Science And Engineering, pages 858-859,Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed byEnglisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Researchand Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRCPress, 1993. Certain of these nucleobases are particularly useful forincreasing the binding affinity of the oligomeric compounds featured inthe invention. These include 5-substituted pyrimidines, 6-azapyrimidinesand N-2, N-6 and 0-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. andLebleu, B., Eds., dsRNA Research and Applications, CRC Press, BocaRaton, 1993, pp. 276-278) and are exemplary base substitutions, evenmore particularly when combined with 2′-O-methoxyethyl sugarmodifications.

Representative U.S. patents that teach the preparation of certain of theabove noted modified nucleobases as well as other modified nucleobasesinclude, but are not limited to, the above noted U.S. Pat. Nos.3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066;5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941;5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887;6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and7,495,088, the entire contents of each of which are hereby incorporatedherein by reference.

The RNA of an iRNA can also be modified to include one or more bicyclicsugar moities. A “bicyclic sugar” is a furanosyl ring modified by thebridging of two atoms. A“bicyclic nucleoside” (“BNA”) is a nucleosidehaving a sugar moiety comprising a bridge connecting two carbon atoms ofthe sugar ring, thereby forming a bicyclic ring system. In certainembodiments, the bridge connects the 4′-carbon and the 2′-carbon of thesugar ring. Thus, in some embodiments an agent of the invention mayinclude one or more locked nucleic acids (LNA). A locked nucleic acid isa nucleotide having a modified ribose moiety in which the ribose moietycomprises an extra bridge connecting the 2′ and 4′ carbons. In otherwords, an LNA is a nucleotide comprising a bicyclic sugar moietycomprising a 4′-CH2-O-2′ bridge. This structure effectively “locks” theribose in the 3′-endo structural conformation. The addition of lockednucleic acids to siRNAs has been shown to increase siRNA stability inserum, and to reduce off-target effects (Elmen, J. et al., (2005)Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol CancTher 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research31(12):3185-3193). Examples of bicyclic nucleosides for use in thepolynucleotides of the invention include without limitation nucleosidescomprising a bridge between the 4′ and the 2′ ribosyl ring atoms. Incertain embodiments, the antisense polynucleotide agents of theinvention include one or more bicyclic nucleosides comprising a 4′ to 2′bridge. Examples of such 4′ to 2′ bridged bicyclic nucleosides, includebut are not limited to 4′-(CH2)-O-2′ (LNA); 4′-(CH2)-S-2′;4′-(CH2)2-O-2′ (ENA); 4′-CH(CH3)-O-2′ (also referred to as “constrainedethyl” or “cEt”) and 4′-CH(CH2OCH3)-O-2′ (and analogs thereof; see,e.g., U.S. Pat. No. 7,399,845); 4′-C(CH3)(CH3)-O-2′ (and analogsthereof; see e.g., U.S. Pat. No. 8,278,283); 4′-CH2-N(OCH3)-2′ (andanalogs thereof; see e.g., U.S. Pat. No. 8,278,425); 4′-CH2-O—N(CH3)-2′(see, e.g., U.S. Patent Publication No. 2004/0171570); 4′-CH2-N(R)—O-2′,wherein R is H, C1-C12 alkyl, or a protecting group (see, e.g., U.S.Pat. No. 7,427,672); 4′-CH2-C(H)(CH3)-2′ (see, e.g., Chattopadhyaya etal., J. Org. Chem., 2009, 74, 118-134); and 4′-CH2-C(═CH2)-2′ (andanalogs thereof; see, e.g., U.S. Pat. No. 8,278,426). The entirecontents of each of the foregoing are hereby incorporated herein byreference.

Additional representative U.S. patents and US Patent Publications thatteach the preparation of locked nucleic acid nucleotides include, butare not limited to, the following: U.S. Pat. Nos. 6,268,490; 6,525,191;6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133;7,084,125; 7,399,845; 7,427,672; 7,569,686; 7,741,457; 8,022,193;8,030,467; 8,278,425; 8,278,426; 8,278,283; US 2008/0039618; and US2009/0012281, the entire contents of each of which are herebyincorporated herein by reference.

Any of the foregoing bicyclic nucleosides can be prepared having one ormore stereochemical sugar configurations including for exampleα-L-ribofuranose and β-D-ribofuranose (see WO 99/14226).

The RNA of an iRNA can also be modified to include one or moreconstrained ethyl nucleotides. As used herein, a “constrained ethylnucleotide” or “cEt” is a locked nucleic acid comprising a bicyclicsugar moiety comprising a 4′-CH(CH3)-0-2′ bridge. In one embodiment, aconstrained ethyl nucleotide is in the S conformation referred to hereinas “S-cEt.”

An iRNA of the invention may also include one or more “conformationallyrestricted nucleotides” (“CRN”). CRN are nucleotide analogs with alinker connecting the C2′ and C4′ carbons of ribose or the C3 and —C5′carbons of ribose. CRN lock the ribose ring into a stable conformationand increase the hybridization affinity to mRNA. The linker is ofsufficient length to place the oxygen in an optimal position forstability and affinity resulting in less ribose ring puckering.

Representative publications that teach the preparation of certain of theabove noted CRN include, but are not limited to, US Patent PublicationNo. 2013/0190383; and PC publication WO 2013/036868, the entire contentsof each of which are hereby incorporated herein by reference.

One or more of the nucleotides of an iRNA of the invention may alsoinclude a hydroxymethyl substituted nucleotide. A “hydroxymethylsubstituted nucleotide” is an acyclic 2′-3′-seco-nucleotide, alsoreferred to as an “unlocked nucleic acid” (“UNA”) modification.

Representative U.S. publications that teach the preparation of UNAinclude, but are not limited to, U.S. Pat. No. 8,314,227; and US PatentPublication Nos. 2013/0096289; 2013/0011922; and 2011/0313020, theentire contents of each of which are hereby incorporated herein byreference.

Potentially stabilizing modifications to the ends of RNA molecules caninclude N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc),N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol(Hyp-NHAc), thymidine-2′-0-deoxythymidine (ether),N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino),2-docosanoyl-uridine-3″-phosphate, inverted base dT(idT) and others.Disclosure of this modification can be found in PCT Publication No. WO2011/005861.

Other modifications of the nucleotides of an iRNA of the inventioninclude a 5′ phosphate or 5′ phosphate mimic, e.g., a 5′-terminalphosphate or phosphate mimic on the antisense strand of an RNAi agent.Suitable phosphate mimics are disclosed in, for example US PatentPublication No. 2012/0157511, the entire contents of which areincorporated herein by reference.

A. Modified iRNAs Comprising Motifs of the Invention

In certain aspects of the invention, the double stranded RNAi agents ofthe invention include chemical modifications as disclosed, for example,in U.S. Provisional Application No. 61/561,710, filed on Nov. 18, 2011,or in PCT/US2012/065691, filed on Nov. 16, 2012, the entire contents ofeach of which are incorporated herein by reference.

More specifically, it has been surprisingly discovered that when thesense strand and antisense strand of the double stranded RNAi agent aremodified to have one or more motifs of three identical modifications onthree consecutive nucleotides at or near the cleavage site of at leastone strand of an RNAi agent, the gene silencing activity of the RNAiagent was superiorly enhanced.

Accordingly, the invention provides double stranded RNAi agents capableof inhibiting the expression of a target gene (i.e., TTR gene) in vivo.The RNAi agent comprises a sense strand and an antisense strand. Eachstrand of the RNAi agent may range from 12-30 nucleotides in length. Forexample, each strand may be between 14-30 nucleotides in length, 17-30nucleotides in length, 25-30 nucleotides in length, 27-30 nucleotides inlength, 17-23 nucleotides in length, 17-21 nucleotides in length, 17-19nucleotides in length, 19-25 nucleotides in length, 19-23 nucleotides inlength, 19-21 nucleotides in length, 21-25 nucleotides in length, or21-23 nucleotides in length.

The sense strand and antisense strand typically form a duplex doublestranded RNA (“dsRNA”), also referred to herein as an “RNAi agent.” Theduplex region of an RNAi agent may be 12-30 nucleotide pairs in length.For example, the duplex region can be between 14-30 nucleotide pairs inlength, 17-30 nucleotide pairs in length, 27-30 nucleotide pairs inlength, 17-23 nucleotide pairs in length, 17-21 nucleotide pairs inlength, 17-19 nucleotide pairs in length, 19-25 nucleotide pairs inlength, 19-23 nucleotide pairs in length, 19-21 nucleotide pairs inlength, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs inlength. In another example, the duplex region is selected from 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides in length.

In one embodiment, the RNAi agent may contain one or more overhangregions and/or capping groups at the 3′-end, 5′-end, or both ends of oneor both strands. The overhang can be 1-6 nucleotides in length, forinstance 2-6 nucleotides in length, 1-5 nucleotides in length, 2-5nucleotides in length, 1-4 nucleotides in length, 2-4 nucleotides inlength, 1-3 nucleotides in length, 2-3 nucleotides in length, or 1-2nucleotides in length. The overhangs can be the result of one strandbeing longer than the other, or the result of two strands of the samelength being staggered. The overhang can form a mismatch with the targetmRNA or it can be complementary to the gene sequences being targeted orcan be another sequence. The first and second strands can also bejoined, e.g., by additional bases to form a hairpin, or by othernon-base linkers.

In one embodiment, the nucleotides in the overhang region of the RNAiagent can each independently be a modified or unmodified nucleotideincluding, but not limited to 2′-sugar modified, such as, 2-F,2′-O-methyl, thymidine (T), 2′-O-methoxyethyl-5-methyluridine (Teo),2′-O-methoxyethyladenosine (Aeo), 2′-O-methoxyethyl-5-methylcytidine(m5Ceo), and any combinations thereof. For example, TI can be anoverhang sequence for either end on either strand. The overhang can forma mismatch with the target mRNA or it can be complementary to the genesequences being targeted or can be another sequence.

The 5′- or 3′-overhangs at the sense strand, antisense strand or bothstrands of the RNAi agent may be phosphorylated. In some embodiments,the overhang region(s) contains two nucleotides having aphosphorothioate between the two nucleotides, where the two nucleotidescan be the same or different. In one embodiment, the overhang is presentat the 3′-end of the sense strand, antisense strand, or both strands. Inone embodiment, this 3′-overhang is present in the antisense strand. Inone embodiment, this 3′-overhang is present in the sense strand.

The RNAi agent may contain only a single overhang, which can strengthenthe interference activity of the RNAi, without affecting its overallstability. For example, the single-stranded overhang may be located atthe 3′-terminal end of the sense strand or, alternatively, at the3′-terminal end of the antisense strand. The RNAi may also have a bluntend, located at the 5′-end of the antisense strand (or the 3′-end of thesense strand) or vice versa. Generally, the antisense strand of the RNAihas a nucleotide overhang at the 3′-end, and the 5′-end is blunt. Whilenot wishing to be bound by theory, the asymmetric blunt end at the5′-end of the antisense strand and 3′-end overhang of the antisensestrand favor the guide strand loading into RISC process.

In one embodiment, the RNAi agent comprises a 21 nucleotide sense strandand a 23 nucleotide antisense strand, wherein the sense strand containsat least one motif of three 2′-F modifications on three consecutivenucleotides at positions 9, 10, 11 from the 5′ end; the antisense strandcontains at least one motif of three 2′-O-methyl modifications on threeconsecutive nucleotides at positions 11, 12, 13 from the 5′ end, whereinone end of the RNAi agent is blunt, while the other end comprises a 2nucleotide overhang. Preferably, the 2 nucleotide overhang is at the3′-end of the antisense strand.

When the 2 nucleotide overhang is at the 3′-end of the antisense strand,there may be two phosphorothioate internucleotide linkages between theterminal three nucleotides, wherein two of the three nucleotides are theoverhang nucleotides, and the third nucleotide is a paired nucleotidenext to the overhang nucleotide. In one embodiment, the RNAi agentadditionally has two phosphorothioate internucleotide linkages betweenthe terminal three nucleotides at both the 5′-end of the sense strandand at the 5′-end of the antisense strand. In one embodiment, everynucleotide in the sense strand and the antisense strand of the RNAiagent, including the nucleotides that are part of the motifs aremodified nucleotides. In one embodiment each residue is independentlymodified with a 2′-O-methyl or 3′-fluoro, e.g., in an alternating motif.In one embodiment, all of the nucleotides of an iRNA of the inventionare modified and the iRNA comprises no more than 8 2′-fluoromodifications (e.g., no more than 7 2′-fluoro modifications, no morethan 6 2′-fluoro modifications, no more than 5 2′-fluoro modifications,no more than 4 2′-fluoro modifications, no more than 3 2′-fluoromodifications, or no more than 2 2′-fluoro modifications) on the sensestrand and no more than 6 2′-fluoro modifications (e.g., no more than 52′-fluoro modifications, no more than 4 2′-fluoro modifications, no morethan 3 2′-fluoro modifications, or no more than 2 2′-fluoromodifications) on the antisense strand. Optionally, the RNAi agentfurther comprises a ligand (preferably GalNAc₃).

In one embodiment, the sense strand of the RNAi agent contains at leastone motif of three identical modifications on three consecutivenucleotides, where one of the motifs occurs at the cleavage site in thesense strand.

In one embodiment, the antisense strand of the RNAi agent can alsocontain at least one motif of three identical modifications on threeconsecutive nucleotides, where one of the motifs occurs at or near thecleavage site in the antisense strand

For an RNAi agent having a duplex region of 17-23 nucleotide in length,the cleavage site of the antisense strand is typically around the 10, 11and 12 positions from the 5′-end. Thus the motifs of three identicalmodifications may occur at the 9, 10, 11 positions; 10, 11, 12positions; 11, 12, 13 positions; 12, 13, 14 positions; or 13, 14, 15positions of the antisense strand, the count starting from the 1^(st)nucleotide from the 5′-end of the antisense strand, or, the countstarting from the 1^(st) paired nucleotide within the duplex region fromthe 5′-end of the antisense strand. The cleavage site in the antisensestrand may also change according to the length of the duplex region ofthe RNAi from the 5′-end.

The sense strand of the RNAi agent may contain at least one motif ofthree identical modifications on three consecutive nucleotides at thecleavage site of the strand; and the antisense strand may have at leastone motif of three identical modifications on three consecutivenucleotides at or near the cleavage site of the strand. When the sensestrand and the antisense strand form a dsRNA duplex, the sense strandand the antisense strand can be so aligned that one motif of the threenucleotides on the sense strand and one motif of the three nucleotideson the antisense strand have at least one nucleotide overlap, i.e., atleast one of the three nucleotides of the motif in the sense strandforms a base pair with at least one of the three nucleotides of themotif in the antisense strand. Alternatively, at least two nucleotidesmay overlap, or all three nucleotides may overlap.

In one embodiment, every nucleotide in the sense strand and antisensestrand of the RNAi agent, including the nucleotides that are part of themotifs, may be modified. Each nucleotide may be modified with the sameor different modification which can include one or more alteration ofone or both of the non-linking phosphate oxygens and/or of one or moreof the linking phosphate oxygens; alteration of a constituent of theribose sugar, e.g., of the 2′ hydroxyl on the ribose sugar; wholesalereplacement of the phosphate moiety with “dephospho” linkers;modification or replacement of a naturally occurring base; andreplacement or modification of the ribose-phosphate backbone.

As nucleic acids are polymers of subunits, many of the modificationsoccur at a position which is repeated within a nucleic acid, e.g., amodification of a base, or a phosphate moiety, or a non-linking O of aphosphate moiety. In some cases the modification will occur at all ofthe subject positions in the nucleic acid but in many cases it will not.By way of example, a modification may only occur at a 3′ or 5′ terminalposition, may only occur in a terminal region, e.g., at a position on aterminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of astrand. A modification may occur in a double strand region, a singlestrand region, or in both. A modification may occur only in the doublestrand region of a RNA or may only occur in a single strand region of aRNA. For example, a phosphorothioate modification at a non-linking Oposition may only occur at one or both termini, may only occur in aterminal region, e.g., at a position on a terminal nucleotide or in thelast 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in doublestrand and single strand regions, particularly at termini. The 5′ end orends can be phosphorylated.

It may be possible, e.g., to enhance stability, to include particularbases in overhangs, or to include modified nucleotides or nucleotidesurrogates, in single strand overhangs, e.g., in a 5′ or 3′ overhang, orin both. For example, it can be desirable to include purine nucleotidesin overhangs. In some embodiments all or some of the bases in a 3′ or 5′overhang may be modified, e.g., with a modification described herein.Modifications can include, e.g., the use of modifications at the 2′position of the ribose sugar with modifications that are known in theart, e.g., the use of deoxyribonucleotides, 2′-deoxy-2′-fluoro (2′-F) or2′-O-methyl modified instead of the ribosugar of the nucleobase, andmodifications in the phosphate group, e.g., phosphorothioatemodifications. Overhangs need not be homologous with the targetsequence.

In one embodiment, each residue of the sense strand and antisense strandis independently modified with LNA, CRN, cET, UNA, HNA, CeNA,2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-deoxy,2′-hydroxyl, or 2′-fluoro. The strands can contain more than onemodification. In one embodiment, each residue of the sense strand andantisense strand is independently modified with 2′-O-methyl or2′-fluoro.

At least two different modifications are typically present on the sensestrand and antisense strand. Those two modifications may be the2′-O-methyl or 2′-fluoro modifications, or others.

In one embodiment, the N_(a) and/or N_(b) comprise modifications of analternating pattern. The term “alternating motif” as used herein refersto a motif having one or more modifications, each modification occurringon alternating nucleotides of one strand. The alternating nucleotide mayrefer to one per every other nucleotide or one per every threenucleotides, or a similar pattern. For example, if A, B and C eachrepresent one type of modification to the nucleotide, the alternatingmotif can be “ABABABABABAB . . . ,” “AABBAABBAABB . . . ,” “AABAABAABAAB. . . ,” “AAABAAABAAAB . . . ,” “AAABBBAAABBB . . . ,” or “ABCABCABCABC. . . ,” etc.

The type of modifications contained in the alternating motif may be thesame or different. For example, if A, B, C, D each represent one type ofmodification on the nucleotide, the alternating pattern, i.e.,modifications on every other nucleotide, may be the same, but each ofthe sense strand or antisense strand can be selected from severalpossibilities of modifications within the alternating motif such as“ABABAB . . . ”, “ACACAC . . . ” “BDBDBD . . . ” or “CDCDCD . . . ,”etc.

In one embodiment, the RNAi agent of the invention comprises themodification pattern for the alternating motif on the sense strandrelative to the modification pattern for the alternating motif on theantisense strand is shifted. The shift may be such that the modifiedgroup of nucleotides of the sense strand corresponds to a differentlymodified group of nucleotides of the antisense strand and vice versa.For example, the sense strand when paired with the antisense strand inthe dsRNA duplex, the alternating motif in the sense strand may startwith “ABABAB” from 5′-3′ of the strand and the alternating motif in theantisense strand may start with “BABABA” from 5′-3′ of the strand withinthe duplex region. As another example, the alternating motif in thesense strand may start with “AABBAABB” from 5′-3′ of the strand and thealternating motif in the antisenese strand may start with “BBAABBAA”from 5′-3′ of the strand within the duplex region, so that there is acomplete or partial shift of the modification patterns between the sensestrand and the antisense strand.

In one embodiment, the RNAi agent comprises the pattern of thealternating motif of 2′-O-methyl modification and 2′-F modification onthe sense strand initially has a shift relative to the pattern of thealternating motif of 2′-O-methyl modification and 2′-F modification onthe antisense strand initially, i.e., the 2′-O-methyl modifiednucleotide on the sense strand base pairs with a 2′-F modifiednucleotide on the antisense strand and vice versa. The 1 position of thesense strand may start with the 2′-F modification, and the 1 position ofthe antisense strand may start with the 2′-O-methyl modification.

The introduction of one or more motifs of three identical modificationson three consecutive nucleotides to the sense strand and/or antisensestrand interrupts the initial modification pattern present in the sensestrand and/or antisense strand. This interruption of the modificationpattern of the sense and/or antisense strand by introducing one or moremotifs of three identical modifications on three consecutive nucleotidesto the sense and/or antisense strand surprisingly enhances the genesilencing activity to the target gene.

In one embodiment, when the motif of three identical modifications onthree consecutive nucleotides is introduced to any of the strands, themodification of the nucleotide next to the motif is a differentmodification than the modification of the motif. For example, theportion of the sequence containing the motif is “ . . . N_(a)YYYN_(b) .. . ,” where “Y” represents the modification of the motif of threeidentical modifications on three consecutive nucleotide, and “N_(a)” and“N_(b)” represent a modification to the nucleotide next to the motif“YYY” that is different than the modification of Y, and where N_(a) andN_(b) can be the same or different modifications. Alternatively, N_(a)and/or N_(b) may be present or absent when there is a wing modificationpresent.

The RNAi agent may further comprise at least one phosphorothioate ormethylphosphonate internucleotide linkage. The phosphorothioate ormethylphosphonate internucleotide linkage modification may occur on anynucleotide of the sense strand or antisense strand or both strands inany position of the strand. For instance, the internucleotide linkagemodification may occur on every nucleotide on the sense strand and/orantisense strand; each internucleotide linkage modification may occur inan alternating pattern on the sense strand and/or antisense strand; orthe sense strand or antisense strand may contain both internucleotidelinkage modifications in an alternating pattern. The alternating patternof the internucleotide linkage modification on the sense strand may bethe same or different from the antisense strand, and the alternatingpattern of the internucleotide linkage modification on the sense strandmay have a shift relative to the alternating pattern of theinternucleotide linkage modification on the antisense strand. In oneembodiment, a double-stranded RNAi agent comprises 6-8phosphorothioateinternucleotide linkages. In one embodiment, the antisense strandcomprises two phosphorothioate internucleotide linkages at the5′-terminus and two phosphorothioate internucleotide linkages at the3′-terminus, and the sense strand comprises at least twophosphorothioate internucleotide linkages at either the 5′-terminus orthe 3′-terminus.

In one embodiment, the RNAi comprises a phosphorothioate ormethylphosphonate internucleotide linkage modification in the overhangregion. For example, the overhang region may contain two nucleotideshaving a phosphorothioate or methylphosphonate internucleotide linkagebetween the two nucleotides. Internucleotide linkage modifications alsomay be made to link the overhang nucleotides with the terminal pairednucleotides within the duplex region. For example, at least 2, 3, 4, orall the overhang nucleotides may be linked through phosphorothioate ormethylphosphonate internucleotide linkage, and optionally, there may beadditional phosphorothioate or methylphosphonate internucleotidelinkages linking the overhang nucleotide with a paired nucleotide thatis next to the overhang nucleotide. For instance, there may be at leasttwo phosphorothioate internucleotide linkages between the terminal threenucleotides, in which two of the three nucleotides are overhangnucleotides, and the third is a paired nucleotide next to the overhangnucleotide. These terminal three nucleotides may be at the 3′-end of theantisense strand, the 3′-end of the sense strand, the 5′-end of theantisense strand, and/or the 5′ end of the antisense strand.

In one embodiment, the 2 nucleotide overhang is at the 3′-end of theantisense strand, and there are two phosphorothioate internucleotidelinkages between the terminal three nucleotides, wherein two of thethree nucleotides are the overhang nucleotides, and the third nucleotideis a paired nucleotide next to the overhang nucleotide. Optionally, theRNAi agent may additionally have two phosphorothioate internucleotidelinkages between the terminal three nucleotides at both the 5′-end ofthe sense strand and at the 5′-end of the antisense strand.

In one embodiment, the RNAi agent comprises mismatch(es) with thetarget, within the duplex, or combinations thereof. The mismatch mayoccur in the overhang region or the duplex region. The base pair may beranked on the basis of their propensity to promote dissociation ormelting (e.g., on the free energy of association or dissociation of aparticular pairing, the simplest approach is to examine the pairs on anindividual pair basis, though next neighbor or similar analysis can alsobe used). In terms of promoting dissociation: A:U is preferred over G:C;G:U is preferred over G:C; and I:C is preferred over G:C (I=inosine).Mismatches, e.g., non-canonical or other than canonical pairings (asdescribed elsewhere herein) are preferred over canonical (A:T, A:U, G:C)pairings; and pairings which include a universal base are preferred overcanonical pairings.

In one embodiment, the RNAi agent comprises at least one of the first 1,2, 3, 4, or 5 base pairs within the duplex regions from the 5′-end ofthe antisense strand independently selected from the group of: A:U, G:U,I:C, and mismatched pairs, e.g., non-canonical or other than canonicalpairings or pairings which include a universal base, to promote thedissociation of the antisense strand at the 5′-end of the duplex.

In one embodiment, the nucleotide at the 1 position within the duplexregion from the 5′-end in the antisense strand is selected from thegroup consisting of A, dA, dU, U, and dT. Alternatively, at least one ofthe first 1, 2 or 3 base pair within the duplex region from the 5′-endof the antisense strand is an AU base pair. For example, the first basepair within the duplex region from the 5′-end of the antisense strand isan AU base pair.

In one embodiment, the sense strand sequence may be represented byformula (I):5′n _(p)-N_(a)—(XXX)_(i)—N_(b)—YYY—N_(b)—(ZZZ)_(j)—N_(a)-n _(q)3′  (I)

wherein:

i and j are each independently 0 or 1;

p and q are each independently 0-6;

each N_(a) independently represents an oligonucleotide sequencecomprising 0-25 modified nucleotides, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b) independently represents an oligonucleotide sequencecomprising 0-10 modified nucleotides;

each n_(p) and n_(q) independently represent an overhang nucleotide;

wherein Nb and Y do not have the same modification; and

XXX, YYY and ZZZ each independently represent one motif of threeidentical modifications on three consecutive nucleotides. Preferably YYYis all 2′-F modified nucleotides.

In one embodiment, the N_(a) and/or N_(b) comprise modifications ofalternating pattern.

In one embodiment, the YYY motif occurs at or near the cleavage site ofthe sense strand. For example, when the RNAi agent has a duplex regionof 17-23 nucleotides in length, the YYY motif can occur at or thevicinity of the cleavage site (e.g.: can occur at positions 6, 7, 8, 7,8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12 or 11, 12, 13) of—the sensestrand, the count starting from the 1^(st) nucleotide, from the 5′-end;or optionally, the count starting at the 1^(st) paired nucleotide withinthe duplex region, from the 5′-end.

In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both iand j are 1. The sense strand can therefore be represented by thefollowing formulas:5′n _(p)-N_(a)—YYY—N_(b)—ZZZ—N_(a)-n _(q)3′  (Ib);5′p-N_(a)—XXX—N_(b)—YYY—N_(a)-n _(q)3′  (Ic); or5′n _(p)-N_(a)—XXX—N_(b)—YYY—N_(b)—ZZZ—N_(a)-n _(q)3′  (Id).

When the sense strand is represented by formula (Ib), N_(b) representsan oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0modified nucleotides. Each N_(a) independently can represent anoligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides.

When the sense strand is represented as formula (Ic), N_(b) representsan oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4,0-2 or 0 modified nucleotides. Each N_(a) can independently represent anoligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides.

When the sense strand is represented as formula (Id), each N_(b)independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Preferably, N_(b) is 0, 1,2, 3, 4, 5 or 6 Each N_(a) can independently represent anoligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides. Each of X, Y and Z may be the same or different from eachother.

In other embodiments, i is 0 and j is 0, and the sense strand may berepresented by the formula:5′p-N_(a)—YYY—N_(a)-n _(q)3′  (Ia).

When the sense strand is represented by formula (Ia), each N_(a)independently can represent an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

In one embodiment, the antisense strand sequence of the RNAi may berepresented by formula (II):5′n _(q′)-N_(a)′—(Z′Z′Z′)_(k)—N_(b)′—Y′Y′Y′—N_(b)′—(X′X′X′)_(l)—N′_(a)-n_(p)′3′  (II)

wherein:

k and l are each independently 0 or 1;

p′ and q′ are each independently 0-6;

each N_(a)′ independently represents an oligonucleotide sequencecomprising 0-25 modified nucleotides, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b)′ independently represents an oligonucleotide sequencecomprising 0-10 modified nucleotides;

each n_(p)′ and n_(q)′ independently represent an overhang nucleotide;

wherein N_(b)′ and Y′ do not have the same modification; and

X′X′X′, Y′Y′Y′ and Z′Z′Z′ each independently represent one motif ofthree identical modifications on three consecutive nucleotides.

In one embodiment, the N_(a)′ and/or N_(b)′ comprise modifications ofalternating pattern.

The Y′Y′Y′ motif occurs at or near the cleavage site of the antisensestrand. For example, when the RNAi agent has a duplex region of 17-23nucleotide in length, the Y′Y′Y′ motif can occur at positions 9, 10, 11;10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisensestrand, with the count starting from the 1^(st) nucleotide, from the5′-end; or optionally, the count starting at the 1^(st) pairednucleotide within the duplex region, from the 5′-end. Preferably, theY′Y′Y′ motif occurs at positions 11, 12, 13.

In one embodiment, Y′Y′Y′ motif is all 2′-OMe modified nucleotides.

In one embodiment, k is 1 and l is 0, or k is 0 and l is 1, or both kand l are 1.

The antisense strand can therefore be represented by the followingformulas:5′n _(q′)-N_(a)′—Z′Z′Z′—N_(b)—Y′Y′Y′—N_(a)′-n _(p′)3′  (IIb);5′n _(q′)-N_(a)′—Y′Y′Y′—N_(b)′—X′X′X′-n _(p′)3′  (IIc); or5′n _(q′)-N_(a)′—Z′Z′Z′—N_(b)′—Y′Y′Y′—N_(b)′—X′X′X′—N_(a)′-n_(p′)3′  (IId).

When the antisense strand is represented by formula (IIb), N_(b)′represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7,0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′ independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides.

When the antisense strand is represented as formula (IIc), N_(b)′represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7,0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′ independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides.

When the antisense strand is represented as formula (IId), each N_(b)′independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides. Preferably, N_(b) is 0, 1, 2, 3, 4,5 or 6.

In other embodiments, k is 0 and 1 is 0 and the antisense strand may berepresented by the formula:5′n _(p′)-N_(a′)—Y′Y′Y′—N_(a′)-n _(q′)3′  (Ia).

When the antisense strand is represented as formula (IIa), each N_(a)′independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

Each of X′, Y′ and Z′ may be the same or different from each other.

Each nucleotide of the sense strand and antisense strand may beindependently modified with LNA, CRN, UNA, cEt, HNA, CeNA,2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-hydroxyl, or2′-fluoro. For example, each nucleotide of the sense strand andantisense strand is independently modified with 2′-O-methyl or2′-fluoro. Each X, Y, Z, X′, Y′ and Z′, in particular, may represent a2′-O-methyl modification or a 2′-fluoro modification.

In one embodiment, the sense strand of the RNAi agent may contain YYYmotif occurring at 9, 10 and 11 positions of the strand when the duplexregion is 21 nt, the count starting from the 1^(st) nucleotide from the5′-end, or optionally, the count starting at the 1^(st) pairednucleotide within the duplex region, from the 5′-end; and Y represents2′-F modification.

In one embodiment the antisense strand may contain Y′Y′Y′ motifoccurring at positions 11, 12, 13 of the strand, the count starting fromthe 1^(st) nucleotide from the 5′-end, or optionally, the count startingat the 1^(st) paired nucleotide within the duplex region, from the5′-end; and Y′ represents 2′-O-methyl modification.

The sense strand represented by any one of the above formulas (Ia),(Ib), (Ic), and (Id) forms a duplex with a antisense strand beingrepresented by any one of formulas (IIa), (IIb), (IIc), and (IId),respectively.

Accordingly, the RNAi agents for use in the methods of the invention maycomprise a sense strand and an antisense strand, each strand having 14to 30 nucleotides, the RNAi duplex represented by formula (III):

(III) sense: 5′n_(p)-N_(a)-(X X X)_(i)-N_(b)-Y Y Y-N_(b)-(Z Z Z)_(j)-N_(a)-n_(q )3′antisense: 3′n_(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′-n_(q)′ 5′

wherein:

i, j, k, and l are each independently 0 or 1;

p, p′, q, and q′ are each independently 0-6;

each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 0-25 modified nucleotides, each sequence comprisingat least two differently modified nucleotides;

each N_(b) and N_(b)′ independently represents an oligonucleotidesequence comprising 0-10 modified nucleotides;

-   -   wherein each n_(p)′, n_(p), n_(q)′, and n_(q), each of which may        or may not be present, independently represents an overhang        nucleotide; and

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently representone motif of three identical modifications on three consecutivenucleotides.

In one embodiment, i is 0 and j is 0; or i is 1 and j is 0; or i is 0and j is 1; or both i and j are 0; or both i and j are 1. In anotherembodiment, k is 0 and l is 0; or k is 1 and l is 0; k is 0 and l is 1;or both k and l are 0; or both k and l are 1.

Exemplary combinations of the sense strand and antisense strand forminga RNAi duplex include the formulas below:5′n _(p)-N_(a)—YYY—N_(a)-n _(q)3′3′n _(p)′-N_(a)′—Y′Y′Y′—N_(a) ′n _(q)′5′   (IIIa)5′n _(p)-N_(a)—YYY—N_(b)—ZZZ—N_(a)-n _(q)3′3′n _(p)′-N_(a)′—Y′Y′Y′—N_(b)′—Z′Z′Z′—N_(a) ′n _(q)′5′   (IIIb)5′n _(p)-N_(a)—XXX—N_(b)—YYY—N_(a)-n _(q)3′3′n _(p)′-N_(a)′—X′X′X′—N_(b)′—Y′Y′Y′—N_(a)′-n _(q)′5′   (IIIc)5′n _(p)-N_(a)—XXX—N_(b)—YYY—N_(b)—ZZZ—N_(a)-n _(q)3′3′n _(p)′-N_(a)′—X′X′X′—N_(b)′—Y′Y′Y′—N_(b)′—Z′Z′Z′—N_(a)-n _(q)′5′  (IIId)5′-N_(a)—YYY—N_(b)-3′3′n _(p)′-N_(a)′—Y′Y′Y′—N_(b)′5′   (IIIe)

When the RNAi agent is represented by formula (IIIa), each N_(a)independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

When the RNAi agent is represented by formula (IIIb), each N_(b)independently represents an oligonucleotide sequence comprising 1-10,1-7, 1-5 or 1-4 modified nucleotides. Each N_(a) independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides.

When the RNAi agent is represented as formula (IIIc), each N_(b), N_(b)′independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

When the RNAi agent is represented as formula (IIId), each N_(b), N_(b)′independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a),N_(a)′ independently represents an oligonucleotide sequence comprising2-20, 2-15, or 2-10 modified nucleotides. Each of N_(a), N_(a)′, N_(b)and N_(b)′ independently comprises modifications of alternating pattern.

When the RNAi agent is represented as formula (IIIe), each N_(a),N_(a)′, N_(b), and N_(b)′ independently represents an oligonucleotidesequence comprising 0-25 nucleotides which are either modified orunmodified or combinations thereof, each sequence comprising at leasttwo differently modified nucleotides.

Each of X, Y and Z in formulas (III), (IIIa), (IIIb), (IIIc), (IIId),and (IIIe) may be the same or different from each other.

When the RNAi agent is represented by formula (III), (IIIa), (IIIb),(IIIc), (IIId), and (IIIe), at least one of the Y nucleotides may form abase pair with one of the Y′ nucleotides. Alternatively, at least two ofthe Y nucleotides form base pairs with the corresponding Y′ nucleotides;or all three of the Y nucleotides all form base pairs with thecorresponding Y′ nucleotides.

When the RNAi agent is represented by formula (IIIb) or (IIId), at leastone of the Z nucleotides may form a base pair with one of the Z′nucleotides. Alternatively, at least two of the Z nucleotides form basepairs with the corresponding Z′ nucleotides; or all three of the Znucleotides all form base pairs with the corresponding Z′ nucleotides.

When the RNAi agent is represented as formula (IIIc) or (IIId), at leastone of the X nucleotides may form a base pair with one of the X′nucleotides. Alternatively, at least two of the X nucleotides form basepairs with the corresponding X′ nucleotides; or all three of the Xnucleotides all form base pairs with the corresponding X′ nucleotides.

In one embodiment, the modification on the Y nucleotide is differentthan the modification on the Y′ nucleotide, the modification on the Znucleotide is different than the modification on the Z′ nucleotide,and/or the modification on the X nucleotide is different than themodification on the X′ nucleotide.

In one embodiment, when the RNAi agent is represented by formula (IIId),the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications. Inanother embodiment, when the RNAi agent is represented by formula(IIId), the N_(a) modifications are 2′-O-methyl or 2′-fluoromodifications and n_(p)′>0 and at least one n_(p)′ is linked to aneighboring nucleotide a via phosphorothioate linkage. In yet anotherembodiment, when the RNAi agent is represented by formula (IIId), theN_(a) modifications are 2′-O-methyl or 2′-fluoro modifications, n_(p)′>0and at least one n_(p)′ is linked to a neighboring nucleotide viaphosphorothioate linkage, and the sense strand is conjugated to one ormore GalNAc derivatives attached through a bivalent or trivalentbranched linker (described below). In another embodiment, when the RNAiagent is represented by formula (IIId), the N_(a) modifications are2′-O-methyl or 2′-fluoro modifications, n_(p)′>0 and at least one n_(p)′is linked to a neighboring nucleotide via phosphorothioate linkage, thesense strand comprises at least one phosphorothioate linkage, and thesense strand is conjugated to one or more GalNAc derivatives attachedthrough a bivalent or trivalent branched linker.

In one embodiment, when the RNAi agent is represented by formula (IIIa),the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications,n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotidevia phosphorothioate linkage, the sense strand comprises at least onephosphorothioate linkage, and the sense strand is conjugated to one ormore GalNAc derivatives attached through a bivalent or trivalentbranched linker.

In one embodiment, two RNAi agents represented by formula (III), (IIIa),(IIIb), (IIIc), (IIId), and (IIIe) are linked to each other at the 5′end, and one or both of the 3′ ends and are optionally conjugated to toa ligand. Each of the agents can target the same gene or two differentgenes; or each of the agents can target same gene at two differenttarget sites.

Various publications describe multimeric RNAi agents that can be used inthe methods of the invention. Such publications include WO2007/091269,U.S. Pat. No. 7,858,769, WO2010/141511, WO2007/117686, WO2009/014887 andWO2011/031520 the entire contents of each of which are herebyincorporated herein by reference.

As described in more detail below, the RNAi agent that containsconjugations of one or more carbohydrate moieties to a RNAi agent canoptimize one or more properties of the RNAi agent. In many cases, thecarbohydrate moiety will be attached to a modified subunit of the RNAiagent. For example, the ribose sugar of one or more ribonucleotidesubunits of a dsRNA agent can be replaced with another moiety, e.g., anon-carbohydrate (preferably cyclic) carrier to which is attached acarbohydrate ligand. A ribonucleotide subunit in which the ribose sugarof the subunit has been so replaced is referred to herein as a ribosereplacement modification subunit (RRMS). A cyclic carrier may be acarbocyclic ring system, i.e., all ring atoms are carbon atoms, or aheterocyclic ring system, i.e., one or more ring atoms may be aheteroatom, e.g., nitrogen, oxygen, sulfur. The cyclic carrier may be amonocyclic ring system, or may contain two or more rings, e.g. fusedrings. The cyclic carrier may be a fully saturated ring system, or itmay contain one or more double bonds.

The ligand may be attached to the polynucleotide via a carrier. Thecarriers include (i) at least one “backbone attachment point,”preferably two “backbone attachment points” and (ii) at least one“tethering attachment point.” A “backbone attachment point” as usedherein refers to a functional group, e.g. a hydroxyl group, orgenerally, a bond available for, and that is suitable for incorporationof the carrier into the backbone, e.g., the phosphate, or modifiedphosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A“tethering attachment point” (TAP) in some embodiments refers to aconstituent ring atom of the cyclic carrier, e.g., a carbon atom or aheteroatom (distinct from an atom which provides a backbone attachmentpoint), that connects a selected moiety. The moiety can be, e.g., acarbohydrate, e.g. monosaccharide, disaccharide, trisaccharide,tetrasaccharide, oligosaccharide and polysaccharide. Optionally, theselected moiety is connected by an intervening tether to the cycliccarrier. Thus, the cyclic carrier will often include a functional group,e.g., an amino group, or generally, provide a bond, that is suitable forincorporation or tethering of another chemical entity, e.g., a ligand tothe constituent ring.

The RNAi agents may be conjugated to a ligand via a carrier, wherein thecarrier can be cyclic group or acyclic group; preferably, the cyclicgroup is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl,imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane,oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl,isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl and anddecalin; preferably, the acyclic group is selected from serinol backboneor diethanolamine backbone.

In certain specific embodiments, the RNAi agent, e.g., for use in themethods of the invention, is an agent selected from the group of agentslisted in any one of Tables 1, 3, 5, 6, and 7. These agents may furthercomprise a ligand.

In certain embodiments, the RNAi agent of the invention is an agentselected from the group consisting of AD-66016, AD-65492, AD-66017, andAD-66018.

IV. IRNAs Conjugated to Ligands

Another modification of the RNA of an iRNA of the invention involveschemically linking to the RNA one or more ligands, moieties orconjugates that enhance the activity, cellular distribution or cellularuptake of the iRNA. Such moieties include but are not limited to lipidmoieties such as a cholesterol moiety (Letsinger et al., Proc. Natl.Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al.,Biorg. Med. Chem. Let., 1994, 4:1053-1060), a thioether, e.g.,beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad Sci., 1992,660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993,3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res.,1992, 20:533-538), an aliphatic chain, e.g., dodecandiol or undecylresidues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanovet al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie,1993, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al.,Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or a polyethyleneglycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995,14:969-973), or adamantane acetic acid (Manoharan et al., TetrahedronLett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim.Biophys. Acta, 1995, 1264:229-237), or an octadecylamine orhexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277:923-937).

In one embodiment, a ligand alters the distribution, targeting orlifetime of an iRNA agent into which it is incorporated. In preferredembodiments a ligand provides an enhanced affinity for a selectedtarget, e.g., molecule, cell or cell type, compartment, e.g., a cellularor organ compartment, tissue, organ or region of the body, as, e.g.,compared to a species absent such a ligand. Preferred ligands will nottake part in duplex pairing in a duplexed nucleic acid.

Ligands can include a naturally occurring substance, such as a protein(e.g., human serum albumin (HSA), low-density lipoprotein (LDL), orglobulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan,inulin, cyclodextrin, N-acetylgalactosamine, or hyaluronic acid); or alipid. The ligand can also be a recombinant or synthetic molecule, suchas a synthetic polymer, e.g., a synthetic polyamino acid. Examples ofpolyamino acids include polyamino acid is a polylysine (PLL), polyL-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydridecopolymer, poly(L-lactide-co-glycolide) copolymer, divinyl ether-maleicanhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA),polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane,poly(2-ethylacrylic acid), N-isopropylacrylamide polymers, orpolyphosphazine. Example of polyamines include: polyethylenimine,polylysine (PLL), spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, cationic lipid, cationic porphyrin,quaternary salt of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, monovalent galactose, N-acetyl-galactosamine,N-acetyl-gulucoseamine multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, vitamin A, biotin, or an RGDpeptide or RGD peptide mimetic. In certain embodiments, ligands includemonovalent or multivalent galactose. In certain embodiments, ligandsinclude cholesterol.

Other examples of ligands include dyes, intercalating agents (e.g.acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins(TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA),lipophilic molecules, e.g., cholesterol, cholic acid, adamantane aceticacid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid,O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g.,antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl,substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin),transport/absorption facilitators (e.g., aspirin, vitamin E, folicacid), synthetic ribonucleases (e.g., imidazole, bisimidazole,histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a hepaticcell. Ligands can also include hormones and hormone receptors. They canalso include non-peptidic species, such as lipids, lectins,carbohydrates, vitamins, cofactors, multivalent lactose, multivalentgalactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalentmannose, or multivalent fucose. The ligand can be, for example, alipopolysaccharide, an activator of p38 MAP kinase, or an activator ofNF-κB.

The ligand can be a substance, e.g., a drug, which can increase theuptake of the iRNA agent into the cell, for example, by disrupting thecell's cytoskeleton, e.g., by disrupting the cell's microtubules,microfilaments, and/or intermediate filaments. The drug can be, forexample, taxon, vincristine, vinblastine, cytochalasin, nocodazole,japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, ormyoservin.

In some embodiments, a ligand attached to an iRNA as described hereinacts as a pharmacokinetic modulator (PK modulator). PK modulatorsinclude lipophiles, bile acids, steroids, phospholipid analogues,peptides, protein binding agents, PEG, vitamins etc. Exemplary PKmodulators include, but are not limited to, cholesterol, fatty acids,cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride,phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotinetc. Oligonucleotides that comprise a number of phosphorothioatelinkages are also known to bind to serum protein, thus shortoligonucleotides, e.g., oligonucleotides of about 5 bases, 10 bases, 15bases or 20 bases, comprising multiple of phosphorothioate linkages inthe backbone are also amenable to the present invention as ligands (e.g.as PK modulating ligands). In addition, aptamers that bind serumcomponents (e.g. serum proteins) are also suitable for use as PKmodulating ligands in the embodiments described herein.

Ligand-conjugated oligonucleotides of the invention may be synthesizedby the use of an oligonucleotide that bears a pendant reactivefunctionality, such as that derived from the attachment of a linkingmolecule onto the oligonucleotide (described below). This reactiveoligonucleotide may be reacted directly with commercially-availableligands, ligands that are synthesized bearing any of a variety ofprotecting groups, or ligands that have a linking moiety attachedthereto.

The oligonucleotides used in the conjugates of the present invention maybe conveniently and routinely made through the well-known technique ofsolid-phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is also known to usesimilar techniques to prepare other oligonucleotides, such as thephosphorothioates and alkylated derivatives.

In the ligand-conjugated oligonucleotides and ligand-molecule bearingsequence-specific linked nucleosides of the present invention, theoligonucleotides and oligonucleosides may be assembled on a suitable DNAsynthesizer utilizing standard nucleotide or nucleoside precursors, ornucleotide or nucleoside conjugate precursors that already bear thelinking moiety, ligand-nucleotide or nucleoside-conjugate precursorsthat already bear the ligand molecule, or non-nucleoside ligand-bearingbuilding blocks.

When using nucleotide-conjugate precursors that already bear a linkingmoiety, the synthesis of the sequence-specific linked nucleosides istypically completed, and the ligand molecule is then reacted with thelinking moiety to form the ligand-conjugated oligonucleotide. In someembodiments, the oligonucleotides or linked nucleosides of the presentinvention are synthesized by an automated synthesizer usingphosphoramidites derived from ligand-nucleoside conjugates in additionto the standard phosphoramidites and non-standard phosphoramidites thatare commercially available and routinely used in oligonucleotidesynthesis.

A. Lipid Conjugates

In one embodiment, the ligand or conjugate is a lipid or lipid-basedmolecule. Such a lipid or lipid-based molecule preferably binds a serumprotein, e.g., human serum albumin (HSA). An HSA binding ligand allowsfor distribution of the conjugate to a target tissue, e.g., a non-kidneytarget tissue of the body. For example, the target tissue can be theliver, including parenchymal cells of the liver. Other molecules thatcan bind HSA can also be used as ligands. For example, naproxen oraspirin can be used. A lipid or lipid-based ligand can (a) increaseresistance to degradation of the conjugate, (b) increase targeting ortransport into a target cell or cell membrane, and/or (c) can be used toadjust binding to a serum protein, e.g., HSA.

A lipid based ligand can be used to inhibit, e.g., control the bindingof the conjugate to a target tissue. For example, a lipid or lipid-basedligand that binds to HSA more strongly will be less likely to betargeted to the kidney and therefore less likely to be cleared from thebody. A lipid or lipid-based ligand that binds to HSA less strongly canbe used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably,it binds HSA with a sufficient affinity such that the conjugate will bepreferably distributed to a non-kidney tissue. However, it is preferredthat the affinity not be so strong that the HSA-ligand binding cannot bereversed.

In another preferred embodiment, the lipid based ligand binds HSA weaklyor not at all, such that the conjugate will be preferably distributed tothe kidney. Other moieties that target to kidney cells can also be usedin place of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which istaken up by a target cell, e.g., a proliferating cell. These areparticularly useful for treating disorders characterized by unwantedcell proliferation, e.g., of the malignant or non-malignant type, e.g.,cancer cells. Exemplary vitamins include vitamin A, E, and K. Otherexemplary vitamins include are B vitamin, e.g., folic acid, B12,riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up bytarget cells such as liver cells. Also included are HSA and low densitylipoprotein (LDL).

B. Cell Permeation Agents

In another aspect, the ligand is a cell-permeation agent, preferably ahelical cell-permeation agent. Preferably, the agent is amphipathic. Anexemplary agent is a peptide such as tat or antennopedia. If the agentis a peptide, it can be modified, including a peptidylmimetic,invertomers, non-peptide or pseudo-peptide linkages, and use of D-aminoacids. The helical agent is preferably an alpha-helical agent, whichpreferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (alsoreferred to herein as an oligopeptidomimetic) is a molecule capable offolding into a defined three-dimensional structure similar to a naturalpeptide. The attachment of peptide and peptidomimetics to iRNA agentscan affect pharmacokinetic distribution of the iRNA, such as byenhancing cellular recognition and absorption. The peptide orpeptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5,10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

A peptide or peptidomimetic can be, for example, a cell permeationpeptide, cationic peptide, amphipathic peptide, or hydrophobic peptide(e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety canbe a dendrimer peptide, constrained peptide or crosslinked peptide. Inanother alternative, the peptide moiety can include a hydrophobicmembrane translocation sequence (MTS). An exemplary hydrophobicMTS-containing peptide is RFGF having the amino acid sequenceAAVALLPAVLLALLAP (SEQ ID NO: 11). An RFGF analogue (e.g., amino acidsequence AALLPVLLAAP (SEQ ID NO: 12) containing a hydrophobic MTS canalso be a targeting moiety. The peptide moiety can be a “delivery”peptide, which can carry large polar molecules including peptides,oligonucleotides, and protein across cell membranes. For example,sequences from the HIV Tat protein (GRKKRRQRRRPPQ) (SEQ ID NO: 13) andthe Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK) (SEQ ID NO: 14)have been found to be capable of functioning as delivery peptides. Apeptide or peptidomimetic can be encoded by a random sequence of DNA,such as a peptide identified from a phage-display library, orone-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature,354:82-84, 1991). Examples of a peptide or peptidomimetic tethered to adsRNA agent via an incorporated monomer unit for cell targeting purposesis an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. Apeptide moiety can range in length from about 5 amino acids to about 40amino acids. The peptide moieties can have a structural modification,such as to increase stability or direct conformational properties. Anyof the structural modifications described below can be utilized.

An RGD peptide for use in the compositions and methods of the inventionmay be linear or cyclic, and may be modified, e.g., glycosylated ormethylated, to facilitate targeting to a specific tissue(s).RGD-containing peptides and peptidiomimemtics may include D-amino acids,as well as synthetic RGD mimics. In addition to RGD, one can use othermoieties that target the integrin ligand. Preferred conjugates of thisligand target PECAM-1 or VEGF.

A “cell permeation peptide” is capable of permeating a cell, e.g., amicrobial cell, such as a bacterial or fungal cell, or a mammalian cell,such as a human cell. A microbial cell-permeating peptide can be, forexample, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), adisulfide bond-containing peptide (e.g., α-defensin, β-defensin orbactenecin), or a peptide containing only one or two dominating aminoacids (e.g., PR-39 or indolicidin). A cell permeation peptide can alsoinclude a nuclear localization signal (NLS). For example, a cellpermeation peptide can be a bipartite amphipathic peptide, such as MPG,which is derived from the fusion peptide domain of HIV-1 gp41 and theNLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.31:2717-2724, 2003).

C. Carbohydrate Conjugates

In some embodiments of the compositions and methods of the invention, aniRNA oligonucleotide further comprises a carbohydrate. The carbohydrateconjugated iRNA are advantageous for the in vivo delivery of nucleicacids, as well as compositions suitable for in vivo therapeutic use, asdescribed herein. As used herein, “carbohydrate” refers to a compoundwhich is either a carbohydrate per se made up of one or moremonosaccharide units having at least 6 carbon atoms (which can belinear, branched or cyclic) with an oxygen, nitrogen or sulfur atombonded to each carbon atom; or a compound having as a part thereof acarbohydrate moiety made up of one or more monosaccharide units eachhaving at least six carbon atoms (which can be linear, branched orcyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbonatom. Representative carbohydrates include the sugars (mono-, di-, tri-and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9monosaccharide units), and polysaccharides such as starches, glycogen,cellulose and polysaccharide gums. Specific monosaccharides include TTRand above (e.g., TTR, C6, C7, or C8) sugars; di- and trisaccharidesinclude sugars having two or three monosaccharide units (e.g., TTR, C6,C7, or C8).

In one embodiment, a carbohydrate conjugate for use in the compositionsand methods of the invention is a monosaccharide. In another embodiment,a carbohydrate conjugate for use in the compositions and methods of theinvention is selected from the group consisting of:

In one embodiment, the monosaccharide is an N-acetylgalactosamine, suchas

Another representative carbohydrate conjugate for use in the embodimentsdescribed herein includes, but is not limited to,

-   -   when one of X or Y is an oligonucleotide, the other is a        hydrogen.

In certain embodiments of the invention, the GalNAc or GalNAc derivativeis attached to an iRNA agent of the invention via a monovalent linker.In some embodiments, the GalNAc or GalNAc derivative is attached to aniRNA agent of the invention via a bivalent linker. In yet otherembodiments of the invention, the GalNAc or GalNAc derivative isattached to an iRNA agent of the invention via a trivalent linker.

In one embodiment, the double stranded RNAi agents of the inventioncomprise one GalNAc or GalNAc derivative attached to the iRNA agent. Inanother embodiment, the double stranded RNAi agents of the inventioncomprise a plurality (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAcderivatives, each independently attached to a plurality of nucleotidesof the double stranded RNAi agent through a plurality of monovalentlinkers.

In some embodiments, for example, when the two strands of an iRNA agentof the invention are part of one larger molecule connected by anuninterrupted chain of nucleotides between the 3′-end of one strand andthe 5′-end of the respective other strand forming a hairpin loopcomprising, a plurality of unpaired nucleotides, each unpairednucleotide within the hairpin loop may independently comprise a GalNAcor GalNAc derivative attached via a monovalent linker. The hairpin loopmay also be formed by an extended overhang in one strand of the duplex.

In some embodiments, the carbohydrate conjugate further comprises one ormore additional ligands as described above, such as, but not limited to,a PK modulator and/or a cell permeation peptide.

Additional carbohydrate conjugates suitable for use in the presentinvention include those described in PCT Publication Nos. WO 2014/179620and WO 2014/179627, the entire contents of each of which areincorporated herein by reference.

D. Linkers

In some embodiments, the conjugate or ligand described herein can beattached to an iRNA oligonucleotide with various linkers that can becleavable or non-cleavable.

The term “linker” or “linking group” means an organic moiety thatconnects two parts of a compound, e.g., covalently attaches two parts ofa compound. Linkers typically comprise a direct bond or an atom such asoxygen or sulfur, a unit such as NR8, C(O), C(O)NH, SO, SO₂, SO₂NH or achain of atoms, such as, but not limited to, substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl,heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl,heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl,heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl,alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl,alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl,alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl,alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl,alkenylheteroarylalkenyl, alkenylheteroarylalkynyl,alkynylheteroarylalkyl, alkynylheteroarylalkenyl,alkynylheteroarylalkynyl, alkylheterocyclylalkyl,alkylheterocyclylalkenyl, alkylhererocyclylalkynyl,alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl,alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl,alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl,alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl,alkynylhereroaryl, which one or more methylenes can be interrupted orterminated by O, S, S(O), SO₂, N(R8), C(O), substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic orsubstituted aliphatic. In one embodiment, the linker is between about1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17,8-17, 6-16, 7-16, or 8-16 atoms.

A cleavable linking group is one which is sufficiently stable outsidethe cell, but which upon entry into a target cell is cleaved to releasethe two parts the linker is holding together. In a preferred embodiment,the cleavable linking group is cleaved at least about 10 times, 20,times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90times or more, or at least about 100 times faster in a target cell orunder a first reference condition (which can, e.g., be selected to mimicor represent intracellular conditions) than in the blood of a subject,or under a second reference condition (which can, e.g., be selected tomimic or represent conditions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH,redox potential or the presence of degradative molecules. Generally,cleavage agents are more prevalent or found at higher levels oractivities inside cells than in serum or blood. Examples of suchdegradative agents include: redox agents which are selected forparticular substrates or which have no substrate specificity, including,e.g., oxidative or reductive enzymes or reductive agents such asmercaptans, present in cells, that can degrade a redox cleavable linkinggroup by reduction; esterases; endosomes or agents that can create anacidic environment, e.g., those that result in a pH of five or lower;enzymes that can hydrolyze or degrade an acid cleavable linking group byacting as a general acid, peptidases (which can be substrate specific),and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptibleto pH. The pH of human serum is 7.4, while the average intracellular pHis slightly lower, ranging from about 7.1-7.3. Endosomes have a moreacidic pH, in the range of 5.5-6.0, and lysosomes have an even moreacidic pH at around 5.0. Some linkers will have a cleavable linkinggroup that is cleaved at a preferred pH, thereby releasing a cationiclipid from the ligand inside the cell, or into the desired compartmentof the cell.

A linker can include a cleavable linking group that is cleavable by aparticular enzyme. The type of cleavable linking group incorporated intoa linker can depend on the cell to be targeted. For example, aliver-targeting ligand can be linked to a cationic lipid through alinker that includes an ester group. Liver cells are rich in esterases,and therefore the linker will be cleaved more efficiently in liver cellsthan in cell types that are not esterase-rich. Other cell-types rich inesterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell typesrich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group canbe evaluated by testing the ability of a degradative agent (orcondition) to cleave the candidate linking group. It will also bedesirable to also test the candidate cleavable linking group for theability to resist cleavage in the blood or when in contact with othernon-target tissue. Thus, one can determine the relative susceptibilityto cleavage between a first and a second condition, where the first isselected to be indicative of cleavage in a target cell and the second isselected to be indicative of cleavage in other tissues or biologicalfluids, e.g., blood or serum. The evaluations can be carried out in cellfree systems, in cells, in cell culture, in organ or tissue culture, orin whole animals. It can be useful to make initial evaluations incell-free or culture conditions and to confirm by further evaluations inwhole animals. In preferred embodiments, useful candidate compounds arecleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, orabout 100 times faster in the cell (or under in vitro conditionsselected to mimic intracellular conditions) as compared to blood orserum (or under in vitro conditions selected to mimic extracellularconditions).

i. Redox Cleavable Linking Groups

In one embodiment, a cleavable linking group is a redox cleavablelinking group that is cleaved upon reduction or oxidation. An example ofreductively cleavable linking group is a disulphide linking group(—S—S—). To determine if a candidate cleavable linking group is asuitable “reductively cleavable linking group,” or for example issuitable for use with a particular iRNA moiety and particular targetingagent one can look to methods described herein. For example, a candidatecan be evaluated by incubation with dithiothreitol (DTT), or otherreducing agent using reagents know in the art, which mimic the rate ofcleavage which would be observed in a cell, e.g., a target cell. Thecandidates can also be evaluated under conditions which are selected tomimic blood or serum conditions. In one, candidate compounds are cleavedby at most about 10% in the blood. In other embodiments, usefulcandidate compounds are degraded at least about 2, 4, 10, 20, 30, 40,50, 60, 70, 80, 90, or about 100 times faster in the cell (or under invitro conditions selected to mimic intracellular conditions) as comparedto blood (or under in vitro conditions selected to mimic extracellularconditions). The rate of cleavage of candidate compounds can bedetermined using standard enzyme kinetics assays under conditions chosento mimic intracellular media and compared to conditions chosen to mimicextracellular media.

ii. Phosphate-Based Cleavable Linking Groups

In another embodiment, a cleavable linker comprises a phosphate-basedcleavable linking group. A phosphate-based cleavable linking group iscleaved by agents that degrade or hydrolyze the phosphate group. Anexample of an agent that cleaves phosphate groups in cells are enzymessuch as phosphatases in cells. Examples of phosphate-based linkinggroups are —O—P(O)(ORk)-O—, —O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—,—S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—,—S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—, —O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—,—S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(S)(Rk)-S—. Preferred embodimentsare —O—P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—,—O—P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—,—O—P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)—O, —S—P(S)(H)—O—,—S—P(O)(H)—S—, —O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—.These candidates can be evaluated using methods analogous to thosedescribed above.

iii. Acid Cleavable Linking Groups

In another embodiment, a cleavable linker comprises an acid cleavablelinking group. An acid cleavable linking group is a linking group thatis cleaved under acidic conditions. In preferred embodiments acidcleavable linking groups are cleaved in an acidic environment with a pHof about 6.5 or lower (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or lower),or by agents such as enzymes that can act as a general acid. In a cell,specific low pH organelles, such as endosomes and lysosomes can providea cleaving environment for acid cleavable linking groups. Examples ofacid cleavable linking groups include but are not limited to hydrazones,esters, and esters of amino acids. Acid cleavable groups can have thegeneral formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is whenthe carbon attached to the oxygen of the ester (the alkoxy group) is anaryl group, substituted alkyl group, or tertiary alkyl group such asdimethyl pentyl or t-butyl. These candidates can be evaluated usingmethods analogous to those described above.

iv. Ester-Based Linking Groups

In another embodiment, a cleavable linker comprises an ester-basedcleavable linking group. An ester-based cleavable linking group iscleaved by enzymes such as esterases and amidases in cells. Examples ofester-based cleavable linking groups include but are not limited toesters of alkylene, alkenylene and alkynylene groups. Ester cleavablelinking groups have the general formula —C(O)O—, or —OC(O)—. Thesecandidates can be evaluated using methods analogous to those describedabove.

v. Peptide-Based Cleaving Groups

In yet another embodiment, a cleavable linker comprises a peptide-basedcleavable linking group. A peptide-based cleavable linking group iscleaved by enzymes such as peptidases and proteases in cells.Peptide-based cleavable linking groups are peptide bonds formed betweenamino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.)and polypeptides. Peptide-based cleavable groups do not include theamide group (—C(O)NH—). The amide group can be formed between anyalkylene, alkenylene or alkynelene. A peptide bond is a special type ofamide bond formed between amino acids to yield peptides and proteins.The peptide based cleavage group is generally limited to the peptidebond (i.e., the amide bond) formed between amino acids yielding peptidesand proteins and does not include the entire amide functional group.Peptide-based cleavable linking groups have the general formula—NHCHRAC(O)NHCHRBC(O)—, where RA and RB are the R groups of the twoadjacent amino acids. These candidates can be evaluated using methodsanalogous to those described above.

In one embodiment, an iRNA of the invention is conjugated to acarbohydrate through a linker. Non-limiting examples of iRNAcarbohydrate conjugates with linkers of the compositions and methods ofthe invention include, but are not limited to,

when one of X or Y is an oligonucleotide, the other is a hydrogen.

In certain embodiments of the compositions and methods of the invention,a ligand is one or more “GalNAc” (N-acetylgalactosamine) derivativesattached through a bivalent or trivalent branched linker.

In one embodiment, a dsRNA of the invention is conjugated to a bivalentor trivalent branched linker selected from the group of structures shownin any of formula (XXXII)-(XXXV):

wherein:q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C represent independentlyfor each occurrence q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5Crepresent independently for each occurrence 0-20 and wherein therepeating unit can be the same or different;P^(2A), P^(2B), P^(3A), P^(3B), P^(4A), P^(4B), P^(5A), P^(5B), P^(5C),T^(2A), T^(2B), T^(3A), T^(3B), T^(4A), T^(4B), T^(4A), T^(5B), T^(5C)are each independently for each occurrence absent, CO, NH, O, S, OC(O),NHC(O), CH₂, CH₂NH or CH₂O;Q^(2A), Q^(2B), Q^(3A), Q^(3B), Q^(4A), Q^(4B), Q^(5A), Q^(5B), Q^(5C)are independently for each occurrence absent, alkylene, substitutedalkylene wherein one or more methylenes can be interrupted or terminatedby one or more of O, S, S(O), SO₂, N(R^(N)), C(R′)═C(R″), C≡C or C(O);R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), R^(4B), R^(5A), R^(5B), R^(5C)are each independently for each occurrence absent, NH, O, S, CH₂, C(O)O,C(O)NH, NHCH(R^(a))C(O), —C(O)—CH(R^(a))—NH—, CO,

or heterocyclyl;

L^(2A), L^(2B), L^(3A), L^(3B), L^(4A), L^(4B), L^(5A), L^(5B) andL^(5C) represent the ligand; i.e. each independently for each occurrencea monosaccharide (such as GalNAc), disaccharide, trisaccharide,tetrasaccharide, oligosaccharide, or polysaccharide; and R^(a) is H oramino acid side chain. Trivalent conjugating GalNAc derivatives areparticularly useful for use with RNAi agents for inhibiting theexpression of a target gene, such as those of formula (XXXVI):

-   -   wherein L^(5A), L^(5B) and L^(5C) represent a monosaccharide,        such as GalNAc derivative.

Examples of suitable bivalent and trivalent branched linker groupsconjugating GalNAc derivatives include, but are not limited to, thestructures recited above as formulas II, VII, XI, X, and XIII.

Representative U.S. patents that teach the preparation of RNA conjugatesinclude, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882;5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717,5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077;5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735;4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335;4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830;5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536;5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203,5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810;5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923;5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931;6,900,297; 7,037,646; 8,106,022, the entire contents of each of whichare hereby incorporated herein by reference.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications can be incorporated in a single compound or even at asingle nucleoside within an iRNA. The present invention also includesiRNA compounds that are chimeric compounds.

“Chimeric” iRNA compounds or “chimeras,” in the context of thisinvention, are iRNA compounds, preferably dsRNAs, which contain two ormore chemically distinct regions, each made up of at least one monomerunit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAstypically contain at least one region wherein the RNA is modified so asto confer upon the iRNA increased resistance to nuclease degradation,increased cellular uptake, and/or increased binding affinity for thetarget nucleic acid. An additional region of the iRNA can serve as asubstrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. Byway of example, RNase H is a cellular endonuclease which cleaves the RNAstrand of an RNA:DNA duplex. Activation of RNase H, therefore, resultsin cleavage of the RNA target, thereby greatly enhancing the efficiencyof iRNA inhibition of gene expression. Consequently, comparable resultscan often be obtained with shorter iRNAs when chimeric dsRNAs are used,compared to phosphorothioate deoxy dsRNAs hybridizing to the same targetregion. Cleavage of the RNA target can be routinely detected by gelelectrophoresis and, if necessary, associated nucleic acid hybridizationtechniques known in the art.

In certain instances, the RNA of an iRNA can be modified by a non-ligandgroup. A number of non-ligand molecules have been conjugated to iRNAs inorder to enhance the activity, cellular distribution or cellular uptakeof the iRNA, and procedures for performing such conjugations areavailable in the scientific literature. Such non-ligand moieties haveincluded lipid moieties, such as cholesterol (Kubo, T. et al., Biochem.Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl.Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg.Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan etal., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain,e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J.,1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk etal., Biochimie, 1993, 75:49), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990,18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al.,Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid(Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety(Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or anoctadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke etal., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative UnitedStates patents that teach the preparation of such RNA conjugates havebeen listed above. Typical conjugation protocols involve the synthesisof an RNAs bearing an aminolinker at one or more positions of thesequence. The amino group is then reacted with the molecule beingconjugated using appropriate coupling or activating reagents. Theconjugation reaction can be performed either with the RNA still bound tothe solid support or following cleavage of the RNA, in solution phase.Purification of the RNA conjugate by HPLC typically affords the pureconjugate.

V. Delivery of an IRNA of the Invention

The delivery of an iRNA of the invention to a cell e.g., a cell within asubject, such as a human subject (e.g., a subject in need thereof, suchas a subject having a disease, disorder or condition associated withTTR) can be achieved in a number of different ways. For example,delivery may be performed by contacting a cell with an iRNA of theinvention either in vitro or in vivo. In vivo delivery may also beperformed directly by administering a composition comprising an iRNA,e.g., a dsRNA, to a subject. Alternatively, in vivo delivery may beperformed indirectly by administering one or more vectors that encodeand direct the expression of the iRNA. These alternatives are discussedfurther below.

In general, any method of delivering a nucleic acid molecule (in vitroor in vivo) can be adapted for use with an iRNA of the invention (seee.g., Akhtar S. and Julian R L. (1992) Trends Cell. Biol. 2(5):139-144and WO94/02595, which are incorporated herein by reference in theirentireties). For in vivo delivery, factors to consider in order todeliver an iRNA molecule include, for example, biological stability ofthe delivered molecule, prevention of non-specific effects, andaccumulation of the delivered molecule in the target tissue. Thenon-specific effects of an iRNA can be minimized by localadministration, for example, by direct injection or implantation into atissue or topically administering the preparation. Local administrationto a treatment site maximizes local concentration of the agent, limitsthe exposure of the agent to systemic tissues that can otherwise beharmed by the agent or that can degrade the agent, and permits a lowertotal dose of the iRNA molecule to be administered. Several studies haveshown successful knockdown of gene products when an iRNA is administeredlocally. For example, intraocular delivery of a VEGF dsRNA byintravitreal injection in cynomolgus monkeys (Tolentino, M J., et al(2004) Retina 24:132-138) and subretinal injections in mice (Reich, SJ., et al (2003) Mol. Vis. 9:210-216) were both shown to preventneovascularization in an experimental model of age-related maculardegeneration. In addition, direct intratumoral injection of a dsRNA inmice reduces tumor volume (Pille, J., et al (2005) Mol. Ther.11:267-274) and can prolong survival of tumor-bearing mice (Kim, W J.,et al (2006) Mol. Ther. 14:343-350; Li, S., et al (2007) Mol. Ther.15:515-523). RNA interference has also shown success with local deliveryto the CNS by direct injection (Dorn, G., et al. (2004) Nucleic Acids32:e49; Tan, P H., et al (2005) Gene Ther. 12:59-66; Makimura, H., et al(2002) BMC Neurosci. 3:18; Shishkina, G T., et al (2004) Neuroscience129:521-528; Thakker, E R., et al (2004) Proc. Natl. Acad. Sci. U.S.A.101:17270-17275; Akaneya, Y., et al (2005) J. Neurophysiol. 93:594-602)and to the lungs by intranasal administration (Howard, K A., et al(2006) Mol. Ther. 14:476-484; Zhang, X., et al (2004) J. Biol. Chem.279:10677-10684; Bitko, V., et al (2005) Nat. Med. 11:50-55). Foradministering an iRNA systemically for the treatment of a disease, theRNA can be modified or alternatively delivered using a drug deliverysystem; both methods act to prevent the rapid degradation of the dsRNAby endo- and exo-nucleases in vivo. Modification of the RNA or thepharmaceutical carrier can also permit targeting of the iRNA compositionto the target tissue and avoid undesirable off-target effects. iRNAmolecules can be modified by chemical conjugation to lipophilic groupssuch as cholesterol to enhance cellular uptake and prevent degradation.For example, an iRNA directed against ApoB conjugated to a lipophiliccholesterol moiety was injected systemically into mice and resulted inknockdown of apoB mRNA in both the liver and jejunum (Soutschek, J., etal (2004) Nature 432:173-178). Conjugation of an iRNA to an aptamer hasbeen shown to inhibit tumor growth and mediate tumor regression in amouse model of prostate cancer (McNamara, J O., et al (2006) Nat.Biotechnol. 24:1005-1015). In an alternative embodiment, the iRNA can bedelivered using drug delivery systems such as a nanoparticle, adendrimer, a polymer, liposomes, or a cationic delivery system.Positively charged cationic delivery systems facilitate binding of aniRNA molecule (negatively charged) and also enhance interactions at thenegatively charged cell membrane to permit efficient uptake of an iRNAby the cell. Cationic lipids, dendrimers, or polymers can either bebound to an iRNA, or induced to form a vesicle or micelle (see e.g., KimS H., et al (2008) Journal of Controlled Release 129(2):107-116) thatencases an iRNA. The formation of vesicles or micelles further preventsdegradation of the iRNA when administered systemically. Methods formaking and administering cationic-iRNA complexes are well within theabilities of one skilled in the art (see e.g., Sorensen, D R., et al(2003) J. Mol. Biol 327:761-766; Verma, U N., et al (2003) Clin. CancerRes. 9:1291-1300; Arnold, A S et al (2007) J. Hypertens. 25:197-205,which are incorporated herein by reference in their entirety). Somenon-limiting examples of drug delivery systems useful for systemicdelivery of iRNAs include DOTAP (Sorensen, D R., et al (2003), supra;Verma, U N., et al (2003), supra), Oligofectamine, “solid nucleic acidlipid particles” (Zimmermann, T S., et al (2006) Nature 441:111-114),cardiolipin (Chien, P Y., et al (2005) Cancer Gene Ther. 12:321-328;Pal, A., et al (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine(Bonnet M E., et al (2008) Pharm. Res. August 16 Epub ahead of print;Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD)peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines(Tomalia, D A., et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., etal (1999) Pharm. Res. 16:1799-1804). In some embodiments, an iRNA formsa complex with cyclodextrin for systemic administration. Methods foradministration and pharmaceutical compositions of iRNAs andcyclodextrins can be found in U.S. Pat. No. 7,427,605, which is hereinincorporated by reference in its entirety.

A. Vector Encoded iRNAs of the Invention

iRNA targeting the TTR gene can be expressed from transcription unitsinserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG.(1996), 12:5-10; Skillern, A., et al., International PCT Publication No.WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, andConrad, U.S. Pat. No. 6,054,299). Expression can be transient (on theorder of hours to weeks) or sustained (weeks to months or longer),depending upon the specific construct used and the target tissue or celltype. These transgenes can be introduced as a linear construct, acircular plasmid, or a viral vector, which can be an integrating ornon-integrating vector. The transgene can also be constructed to permitit to be inherited as an extrachromosomal plasmid (Gassmann, et al.,Proc. Natl. Acad. Sci. USA (1995) 92:1292).

The individual strand or strands of an iRNA can be transcribed from apromoter on an expression vector. Where two separate strands are to beexpressed to generate, for example, a dsRNA, two separate expressionvectors can be co-introduced (e.g., by transfection or infection) into atarget cell. Alternatively each individual strand of a dsRNA can betranscribed by promoters both of which are located on the sameexpression plasmid. In one embodiment, a dsRNA is expressed as invertedrepeat polynucleotides joined by a linker polynucleotide sequence suchthat the dsRNA has a stem and loop structure.

iRNA expression vectors are generally DNA plasmids or viral vectors.Expression vectors compatible with eukaryotic cells, preferably thosecompatible with vertebrate cells, can be used to produce recombinantconstructs for the expression of an iRNA as described herein. Eukaryoticcell expression vectors are well known in the art and are available froma number of commercial sources. Typically, such vectors are providedcontaining convenient restriction sites for insertion of the desirednucleic acid segment. Delivery of iRNA expressing vectors can besystemic, such as by intravenous or intramuscular administration, byadministration to target cells ex-planted from the patient followed byreintroduction into the patient, or by any other means that allows forintroduction into a desired target cell.

iRNA expression plasmids can be transfected into target cells as acomplex with cationic lipid carriers (e.g., Oligofectamine) ornon-cationic lipid-based carriers (e.g., Transit-TKO™). Multiple lipidtransfections for iRNA-mediated knockdowns targeting different regionsof a target RNA over a period of a week or more are also contemplated bythe invention. Successful introduction of vectors into host cells can bemonitored using various known methods. For example, transienttransfection can be signaled with a reporter, such as a fluorescentmarker, such as Green Fluorescent Protein (GFP). Stable transfection ofcells ex vivo can be ensured using markers that provide the transfectedcell with resistance to specific environmental factors (e.g.,antibiotics and drugs), such as hygromycin B resistance.

Viral vector systems which can be utilized with the methods andcompositions described herein include, but are not limited to, (a)adenovirus vectors; (b) retrovirus vectors, including but not limited tolentiviral vectors, moloney murine leukemia virus, etc.; (c)adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h)picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g.,vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) ahelper-dependent or gutless adenovirus. Replication-defective virusescan also be advantageous. Different vectors will or will not becomeincorporated into the cells' genome. The constructs can include viralsequences for transfection, if desired. Alternatively, the construct canbe incorporated into vectors capable of episomal replication, e.g. EPVand EBV vectors. Constructs for the recombinant expression of an iRNAwill generally require regulatory elements, e.g., promoters, enhancers,etc., to ensure the expression of the iRNA in target cells. Otheraspects to consider for vectors and constructs are further describedbelow.

Vectors useful for the delivery of an iRNA will include regulatoryelements (promoter, enhancer, etc.) sufficient for expression of theiRNA in the desired target cell or tissue. The regulatory elements canbe chosen to provide either constitutive or regulated/inducibleexpression.

Expression of the iRNA can be precisely regulated, for example, by usingan inducible regulatory sequence that is sensitive to certainphysiological regulators, e.g., circulating glucose levels, or hormones(Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expressionsystems, suitable for the control of dsRNA expression in cells or inmammals include, for example, regulation by ecdysone, by estrogen,progesterone, tetracycline, chemical inducers of dimerization, andisopropyl-beta-D1-thiogalactopyranoside (IPTG). A person skilled in theart would be able to choose the appropriate regulatory/promoter sequencebased on the intended use of the iRNA transgene.

Viral vectors that contain nucleic acid sequences encoding an iRNA canbe used. For example, a retroviral vector can be used (see Miller etal., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectorscontain the components necessary for the correct packaging of the viralgenome and integration into the host cell DNA. The nucleic acidsequences encoding an iRNA are cloned into one or more vectors, whichfacilitate delivery of the nucleic acid into a patient. More detailabout retroviral vectors can be found, for example, in Boesen et al.,Biotherapy 6:291-302 (1994), which describes the use of a retroviralvector to deliver the mdr1 gene to hematopoietic stem cells in order tomake the stem cells more resistant to chemotherapy. Other referencesillustrating the use of retroviral vectors in gene therapy are: Cloweset al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141(1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel.3:110-114 (1993). Lentiviral vectors contemplated for use include, forexample, the HIV based vectors described in U.S. Pat. Nos. 6,143,520;5,665,557; and 5,981,276, which are herein incorporated by reference.

Adenoviruses are also contemplated for use in delivery of iRNAs of theinvention. Adenoviruses are especially attractive vehicles, e.g., fordelivering genes to respiratory epithelia. Adenoviruses naturally infectrespiratory epithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, CurrentOpinion in Genetics and Development 3:499-503 (1993) present a review ofadenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10(1994) demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al.,Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992);Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT PublicationWO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). A suitableAV vector for expressing an iRNA featured in the invention, a method forconstructing the recombinant AV vector, and a method for delivering thevector into target cells, are described in Xia H et al. (2002), Nat.Biotech. 20: 1006-1010.

Adeno-associated virus (AAV) vectors may also be used to delivery aniRNA of the invention (Walsh et al., Proc. Soc. Exp. Biol. Med.204:289-300 (1993); U.S. Pat. No. 5,436,146). In one embodiment, theiRNA can be expressed as two separate, complementary single-stranded RNAmolecules from a recombinant AAV vector having, for example, either theU6 or HI RNA promoters, or the cytomegalovirus (CMV) promoter. SuitableAAV vectors for expressing the dsRNA featured in the invention, methodsfor constructing the recombinant AV vector, and methods for deliveringthe vectors into target cells are described in Samulski R et al. (1987),J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol, 70:520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat.Nos. 5,252,479; 5,139,941; International Patent Application No. WO94/13788; and International Patent Application No. WO 93/24641, theentire disclosures of which are herein incorporated by reference.

Another viral vector suitable for delivery of an iRNA of the inventionis a pox virus such as a vaccinia virus, for example an attenuatedvaccinia such as Modified Virus Ankara (MVA) or NYVAC, an avipox such asfowl pox or canary pox.

The tropism of viral vectors can be modified by pseudotyping the vectorswith envelope proteins or other surface antigens from other viruses, orby substituting different viral capsid proteins, as appropriate. Forexample, lentiviral vectors can be pseudotyped with surface proteinsfrom vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and thelike. AAV vectors can be made to target different cells by engineeringthe vectors to express different capsid protein serotypes; see, e.g.,Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosureof which is herein incorporated by reference.

The pharmaceutical preparation of a vector can include the vector in anacceptable diluent, or can include a slow release matrix in which thegene delivery vehicle is imbedded. Alternatively, where the completegene delivery vector can be produced intact from recombinant cells,e.g., retroviral vectors, the pharmaceutical preparation can include oneor more cells which produce the gene delivery system.

VI. Pharmaceutical Compositions of the Invention

The present invention also includes pharmaceutical compositions andformulations which include the iRNAs of the invention. In oneembodiment, provided herein are pharmaceutical compositions containingan iRNA, as described herein, and a pharmaceutically acceptable carrier.The pharmaceutical compositions containing the iRNA are useful fortreating a disease or disorder associated with the expression oractivity of a TTR gene. Such pharmaceutical compositions are formulatedbased on the mode of delivery. One example is compositions that areformulated for systemic administration via parenteral delivery, e.g., bysubcutaneous (SC) or intravenous (IV) delivery. Another example iscompositions that are formulated for direct delivery into the brainparenchyma, e.g., by infusion into the brain, such as by continuous pumpinfusion. The pharmaceutical compositions of the invention may beadministered in dosages sufficient to inhibit expression of a TTR gene.In one embodiment, the iRNA agents of the invention, e.g., a dsRNAagent, is formulated for subcutaneous administration in apharmaceutically acceptable carrier

The pharmaceutical composition can be administered by intravenousinfusion over a period of time, such as over a 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, and 21, 22, 23, 24, or about a 25minute period. The administration may be repeated, for example, on aregular basis, such as weekly, biweekly (i.e., every two weeks) for onemonth, two months, three months, four months or longer. Administrationmay also be repeated, for example, on a monthly basis, or on aquartlerly basis, e.g., approximately every 12 weeks. After an initialtreatment regimen, the treatments can be administered on a less frequentbasis. For example, after administration weekly or biweekly for threemonths, administration can be repeated once per month, for six months ora year or longer.

The pharmaceutical composition can be administered once daily, or theiRNA can be administered as two, three, or more sub-doses at appropriateintervals throughout the day or even using continuous infusion ordelivery through a controlled release formulation. In that case, theiRNA contained in each sub-dose must be correspondingly smaller in orderto achieve the total daily dosage. The dosage unit can also becompounded for delivery over several days, e.g., using a conventionalsustained release formulation which provides sustained release of theiRNA over a several day period. Sustained release formulations are wellknown in the art and are particularly useful for delivery of agents at aparticular site, such as could be used with the agents of the presentinvention. In this embodiment, the dosage unit contains a correspondingmultiple of the daily dose.

In other embodiments, a single dose of the pharmaceutical compositionscan be long lasting, such that subsequent doses are administered at notmore than 3, 4, or 5 day intervals, at not more than 1, 2, 3, or 4 weekintervals, or at not more than 9, 10, 11, or 12 week intervals. In someembodiments of the invention, a single dose of the pharmaceuticalcompositions of the invention is administered once per week. In otherembodiments of the invention, a single dose of the pharmaceuticalcompositions of the invention is administered bi-monthly. In otherembodiments, a single dose of the pharmaceutical compositions of theinvention is administered monthly. In still other embodiments, a singledose of the pharmaceutical compositions of the invention is administeredquarterly.

The skilled artisan will appreciate that certain factors can influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the individual iRNAs encompassed by the inventioncan be made using conventional methodologies or on the basis of in vivotesting using an appropriate animal model, as described elsewhereherein.

The pharmaceutical compositions of the present invention can beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration can be topical (e.g., by a transdermal patch), pulmonary,e.g., by inhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal, oral orparenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; subdermal, e.g., via an implanted device; or intracranial,e.g., by intraparenchymal, intrathecal or intraventricular,administration.

The iRNA can be delivered in a manner to target a particular tissue,such as the liver (e.g., the hepatocytes of the liver).

Pharmaceutical compositions and formulations for topical administrationcan include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like can be necessary or desirable. Coated condoms, gloves and thelike can also be useful. Suitable topical formulations include those inwhich the iRNAs featured in the invention are in admixture with atopical delivery agent such as lipids, liposomes, fatty acids, fattyacid esters, steroids, chelating agents and surfactants. Suitable lipidsand liposomes include neutral (e.g., dioleoylphosphatidyl DOPEethanolamine, dimyristoylphosphatidyl choline DMPC,distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidylglycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAPand dioleoylphosphatidyl ethanolamine DOTMA). iRNAs featured in theinvention can be encapsulated within liposomes or can form complexesthereto, in particular to cationic liposomes. Alternatively, iRNAs canbe complexed to lipids, in particular to cationic lipids. Suitable fattyacids and esters include but are not limited to arachidonic acid, oleicacid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristicacid, palmitic acid, stearic acid, linoleic acid, linolenic acid,dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or aC₁₋₂₀ alkyl ester (e.g., isopropylmyristate IPM), monoglyceride,diglyceride or pharmaceutically acceptable salt thereof). Topicalformulations are described in detail in U.S. Pat. No. 6,747,014, whichis incorporated herein by reference.

A. iRNA Formulations Comprising Membranous Molecular Assemblies

An iRNA for use in the compositions and methods of the invention can beformulated for delivery in a membranous molecular assembly, e.g., aliposome or a micelle. As used herein, the term “liposome” refers to avesicle composed of amphiphilic lipids arranged in at least one bilayer,e.g., one bilayer or a plurality of bilayers. Liposomes includeunilamellar and multilamellar vesicles that have a membrane formed froma lipophilic material and an aqueous interior. The aqueous portioncontains the iRNA composition. The lipophilic material isolates theaqueous interior from an aqueous exterior, which typically does notinclude the iRNA composition, although in some examples, it may.Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomal bilayer fuses with bilayer of the cellular membranes. Asthe merging of the liposome and cell progresses, the internal aqueouscontents that include the iRNA are delivered into the cell where theiRNA can specifically bind to a target RNA and can mediate iRNA. In somecases the liposomes are also specifically targeted, e.g., to direct theiRNA to particular cell types.

A liposome containing an iRNA agent can be prepared by a variety ofmethods. In one example, the lipid component of a liposome is dissolvedin a detergent so that micelles are formed with the lipid component. Forexample, the lipid component can be an amphipathic cationic lipid orlipid conjugate. The detergent can have a high critical micelleconcentration and may be nonionic. Exemplary detergents include cholate,CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The iRNAagent preparation is then added to the micelles that include the lipidcomponent. The cationic groups on the lipid interact with the iRNA agentand condense around the iRNA agent to form a liposome. Aftercondensation, the detergent is removed, e.g., by dialysis, to yield aliposomal preparation of iRNA agent.

If necessary a carrier compound that assists in condensation can beadded during the condensation reaction, e.g., by controlled addition.For example, the carrier compound can be a polymer other than a nucleicacid (e.g., spermine or spermidine). pH can also adjusted to favorcondensation.

Methods for producing stable polynucleotide delivery vehicles, whichincorporate a polynucleotide/cationic lipid complex as structuralcomponents of the delivery vehicle, are further described in, e.g., WO96/37194, the entire contents of which are incorporated herein byreference. Liposome formation can also include one or more aspects ofexemplary methods described in Felgner, P. L. et al., Proc. Natl. Acad.Sci., USA 8:7413-7417, 1987; U.S. Pat. Nos. 4,897,355; 5,171,678;Bangham, et al. M. Mol. Biol. 23:238, 1965; Olson, et al. Biochim.Biophys. Acta 557:9, 1979; Szoka, et al. Proc. Natl. Acad. Sci. 75:4194, 1978; Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984; Kim, etal. Biochim. Biophys. Acta 728:339, 1983; and Fukunaga, et al.Endocrinol. 115:757, 1984. Commonly used techniques for preparing lipidaggregates of appropriate size for use as delivery vehicles includesonication and freeze-thaw plus extrusion (see, e.g., Mayer, et al.Biochim. Biophys. Acta 858:161, 1986). Microfluidization can be usedwhen consistently small (50 to 200 nm) and relatively uniform aggregatesare desired (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984). Thesemethods are readily adapted to packaging iRNA agent preparations intoliposomes.

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes which interact with the negatively charged nucleicacid molecules to form a stable complex. The positively charged nucleicacid/liposome complex binds to the negatively charged cell surface andis internalized in an endosome. Due to the acidic pH within theendosome, the liposomes are ruptured, releasing their contents into thecell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147,980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap nucleicacids rather than complex with it. Since both the nucleic acid and thelipid are similarly charged, repulsion rather than complex formationoccurs. Nevertheless, some nucleic acid is entrapped within the aqueousinterior of these liposomes. pH-sensitive liposomes have been used todeliver nucleic acids encoding the thymidine kinase gene to cellmonolayers in culture. Expression of the exogenous gene was detected inthe target cells (Zhou et al., Journal of Controlled Release, 1992, 19,269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Examples of other methods to introduce liposomes into cells in vitro andin vivo include U.S. Pat. Nos. 5,283,185; 5,171,678; WO 94/00569; WO93/24640; WO 91/16024; Felgner, J. Biol. Chem. 269:2550, 1994; Nabel,Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human Gene Ther. 3:649,1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J. 11:417, 1992.

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporine A into different layers ofthe skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4(6) 466).

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome (A) comprisesone or more glycolipids, such as monosialoganglioside G_(M1), or (B) isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. While not wishing to be bound by anyparticular theory, it is thought in the art that, at least forsterically stabilized liposomes containing gangliosides, sphingomyelin,or PEG-derivatized lipids, the enhanced circulation half-life of thesesterically stabilized liposomes derives from a reduced uptake into cellsof the reticuloendothelial system (RES) (Allen et al., FEBS Letters,1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in theart. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside G_(M1), galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al).

In one embodiment, cationic liposomes are used. Cationic liposomespossess the advantage of being able to fuse to the cell membrane.Non-cationic liposomes, although not able to fuse as efficiently withthe plasma membrane, are taken up by macrophages in vivo and can be usedto deliver iRNA agents to macrophages.

Further advantages of liposomes include: liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated iRNA agents in their internal compartments frommetabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms,”Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Importantconsiderations in the preparation of liposome formulations are the lipidsurface charge, vesicle size and the aqueous volume of the liposomes.

A positively charged synthetic cationic lipid,N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA)can be used to form small liposomes that interact spontaneously withnucleic acid to form lipid-nucleic acid complexes which are capable offusing with the negatively charged lipids of the cell membranes oftissue culture cells, resulting in delivery of iRNA agent (see, e.g.,Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 andU.S. Pat. No. 4,897,355 for a description of DOTMA and its use withDNA).

A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP)can be used in combination with a phospholipid to form DNA-complexingvesicles. Lipofectin™ Bethesda Research Laboratories, Gaithersburg, Md.)is an effective agent for the delivery of highly anionic nucleic acidsinto living tissue culture cells that comprise positively charged DOTMAliposomes which interact spontaneously with negatively chargedpolynucleotides to form complexes. When enough positively chargedliposomes are used, the net charge on the resulting complexes is alsopositive. Positively charged complexes prepared in this wayspontaneously attach to negatively charged cell surfaces, fuse with theplasma membrane, and efficiently deliver functional nucleic acids into,for example, tissue culture cells. Another commercially availablecationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane(“DOTAP”) (Boehringer Mannheim, Indianapolis, Ind.) differs from DOTMAin that the oleoyl moieties are linked by ester, rather than etherlinkages.

Other reported cationic lipid compounds include those that have beenconjugated to a variety of moieties including, for example,carboxyspermine which has been conjugated to one of two types of lipidsand includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide(“DOGS”) (Transfectam™, Promega, Madison, Wis.) anddipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”)(see, e.g., U.S. Pat. No. 5,171,678).

Another cationic lipid conjugate includes derivatization of the lipidwith cholesterol (“DC-Chol”) which has been formulated into liposomes incombination with DOPE (See, Gao, X. and Huang, L., Biochim. Biophys.Res. Commun. 179:280, 1991). Lipopolylysine, made by conjugatingpolylysine to DOPE, has been reported to be effective for transfectionin the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta1065:8, 1991). For certain cell lines, these liposomes containingconjugated cationic lipids, are said to exhibit lower toxicity andprovide more efficient transfection than the DOTMA-containingcompositions. Other commercially available cationic lipid productsinclude DMRIE and DMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine(DOSPA) (Life Technology, Inc., Gaithersburg, Md.). Other cationiclipids suitable for the delivery of oligonucleotides are described in WO98/39359 and WO 96/37194.

Liposomal formulations are particularly suited for topicaladministration, liposomes present several advantages over otherformulations. Such advantages include reduced side effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer iRNA agent into the skin. In some implementations,liposomes are used for delivering iRNA agent to epidermal cells and alsoto enhance the penetration of iRNA agent into dermal tissues, e.g., intoskin. For example, the liposomes can be applied topically. Topicaldelivery of drugs formulated as liposomes to the skin has beendocumented (see, e.g., Weiner et al., Journal of Drug Targeting, 1992,vol. 2, 405-410 and du Plessis et al., Antiviral Research, 18, 1992,259-265; Mannino, R. J. and Fould-Fogerite, S., Biotechniques 6:682-690,1988; Itani, T. et al. Gene 56:267-276. 1987; Nicolau, C. et al. Meth.Enz 149:157-176, 1987; Straubinger, R. M. and Papahadjopoulos, D. Meth.Enz. 101:512-527, 1983; Wang, C. Y. and Huang, L., Proc. Natl. Acad.Sci. USA 84:7851-7855, 1987).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver a drug into the dermis of mouse skin. Such formulationswith iRNA agent are useful for treating a dermatological disorder.

Liposomes that include iRNA can be made highly deformable. Suchdeformability can enable the liposomes to penetrate through pore thatare smaller than the average radius of the liposome. For example,transfersomes are a type of deformable liposomes. Transferosomes can bemade by adding surface edge activators, usually surfactants, to astandard liposomal composition. Transfersomes that include iRNA agentcan be delivered, for example, subcutaneously by infection in order todeliver iRNA agent to keratinocytes in the skin. In order to crossintact mammalian skin, lipid vesicles must pass through a series of finepores, each with a diameter less than 50 nm, under the influence of asuitable transdermal gradient. In addition, due to the lipid properties,these transferosomes can be self-optimizing (adaptive to the shape ofpores, e.g., in the skin), self-repairing, and can frequently reachtheir targets without fragmenting, and often self-loading.

Other formulations amenable to the present invention are described inPCT Publication No. WO 2008/042973, the entire contents of which areincorporated herein by reference.

Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes can be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g., they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, in“Pharmaceutical Dosage Forms”, Marcel Dekker, Inc., New York, N.Y.,1988, p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in “Pharmaceutical Dosage Forms”, MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

The iRNA for use in the methods of the invention can also be provided asmicellar formulations. “Micelles” are defined herein as a particulartype of molecular assembly in which amphipathic molecules are arrangedin a spherical structure such that all the hydrophobic portions of themolecules are directed inward, leaving the hydrophilic portions incontact with the surrounding aqueous phase. The converse arrangementexists if the environment is hydrophobic.

A mixed micellar formulation suitable for delivery through transdermalmembranes may be prepared by mixing an aqueous solution of the siRNAcomposition, an alkali metal C₈ to C₂₂ alkyl sulphate, and a micelleforming compounds. Exemplary micelle forming compounds include lecithin,hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid,glycolic acid, lactic acid, chamomile extract, cucumber extract, oleicacid, linoleic acid, linolenic acid, monoolein, monooleates,monolaurates, borage oil, evening of primrose oil, menthol, trihydroxyoxo cholanyl glycine and pharmaceutically acceptable salts thereof,glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethyleneethers and analogues thereof, polidocanol alkyl ethers and analoguesthereof, chenodeoxycholate, deoxycholate, and mixtures thereof. Themicelle forming compounds may be added at the same time or afteraddition of the alkali metal alkyl sulphate. Mixed micelles will formwith substantially any kind of mixing of the ingredients but vigorousmixing in order to provide smaller size micelles.

In one method a first micellar composition is prepared which containsthe siRNA composition and at least the alkali metal alkyl sulphate. Thefirst micellar composition is then mixed with at least three micelleforming compounds to form a mixed micellar composition. In anothermethod, the micellar composition is prepared by mixing the siRNAcomposition, the alkali metal alkyl sulphate and at least one of themicelle forming compounds, followed by addition of the remaining micelleforming compounds, with vigorous mixing.

Phenol and/or m-cresol may be added to the mixed micellar composition tostabilize the formulation and protect against bacterial growth.Alternatively, phenol and/or m-cresol may be added with the micelleforming ingredients. An isotonic agent such as glycerin may also beadded after formation of the mixed micellar composition.

For delivery of the micellar formulation as a spray, the formulation canbe put into an aerosol dispenser and the dispenser is charged with apropellant. The propellant, which is under pressure, is in liquid formin the dispenser. The ratios of the ingredients are adjusted so that theaqueous and propellant phases become one, i.e., there is one phase. Ifthere are two phases, it is necessary to shake the dispenser prior todispensing a portion of the contents, e.g., through a metered valve. Thedispensed dose of pharmaceutical agent is propelled from the meteredvalve in a fine spray.

Propellants may include hydrogen-containing chlorofluorocarbons,hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether. Incertain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) may be used.

The specific concentrations of the essential ingredients can bedetermined by relatively straightforward experimentation. For absorptionthrough the oral cavities, it is often desirable to increase, e.g., atleast double or triple, the dosage for through injection oradministration through the gastrointestinal tract.

B. Lipid Particles

iRNAs, e.g., dsRNAs of in the invention may be fully encapsulated in alipid formulation, e.g., a LNP, or other nucleic acid-lipid particle.

As used herein, the term “LNP” refers to a stable nucleic acid-lipidparticle. LNPs typically contain a cationic lipid, a non-cationic lipid,and a lipid that prevents aggregation of the particle (e.g., a PEG-lipidconjugate). LNPs are extremely useful for systemic applications, as theyexhibit extended circulation lifetimes following intravenous (i.v.)injection and accumulate at distal sites (e.g., sites physicallyseparated from the administration site). LNPs include “pSPLP,” whichinclude an encapsulated condensing agent-nucleic acid complex as setforth in PCT Publication No. WO 00/03683. The particles of the presentinvention typically have a mean diameter of about 50 nm to about 150 nm,more typically about 60 nm to about 130 nm, more typically about 70 nmto about 110 nm, most typically about 70 nm to about 90 nm, and aresubstantially nontoxic. In addition, the nucleic acids when present inthe nucleic acid-lipid particles of the present invention are resistantin aqueous solution to degradation with a nuclease. Nucleic acid-lipidparticles and their method of preparation are disclosed in, e.g., U.S.Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; U.S.Publication No. 2010/0324120 and PCT Publication No. WO 96/40964.

In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g.,lipid to dsRNA ratio) will be in the range of from about 1:1 to about50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, fromabout 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 toabout 9:1. Ranges intermediate to the above recited ranges are alsocontemplated to be part of the invention.

The cationic lipid can be, for example, N,N-dioleyl-N,N-dimethylammoniumchloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N—(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),N—(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA),1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl),1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl),1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-Dioleylamino)-1,2-propanedio (DOAP),1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA),2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) oranalogs thereof,(3aR,5s,6aS)—N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine(ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate (MC3),1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol(Tech G1), or a mixture thereof. The cationic lipid can comprise fromabout 20 mol % to about 50 mol % or about 40 mol % of the total lipidpresent in the particle.

In another embodiment, the compound2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used toprepare lipid-siRNA nanoparticles. Synthesis of2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S.provisional patent application No. 61/107,998 filed on Oct. 23, 2008,which is herein incorporated by reference.

In one embodiment, the lipid-siRNA particle includes 40% 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40%Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of63.0±20 nm and a 0.027 siRNA/Lipid Ratio.

The ionizable/non-cationic lipid can be an anionic lipid or a neutrallipid including, but not limited to, distearoylphosphatidylcholine(DSPC), dioleoylphosphatidylcholine (DOPC),dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol(DOPG), dipalmitoylphosphatidylglycerol (DPPG),dioleoyl-phosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoylphosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or amixture thereof. The non-cationic lipid can be from about 5 mol % toabout 90 mol %, about 10 mol %, or about 58 mol % if cholesterol isincluded, of the total lipid present in the particle.

The conjugated lipid that inhibits aggregation of particles can be, forexample, a polyethyleneglycol (PEG)-lipid including, without limitation,a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), aPEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. ThePEG-DAA conjugate can be, for example, a PEG-dilauryloxypropyl (Ci₂), aPEG-dimyristyloxypropyl (Ci₄), a PEG-dipalmityloxypropyl (Ci₆), or aPEG-distearyloxypropyl (C]₈). The conjugated lipid that preventsaggregation of particles can be from 0 mol % to about 20 mol % or about2 mol % of the total lipid present in the particle.

In some embodiments, the nucleic acid-lipid particle further includescholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol %of the total lipid present in the particle.

In one embodiment, the lipidoid ND98.4HCl (MW 1487) (see U.S. patentapplication Ser. No. 12/056,230, filed Mar. 26, 2008, which isincorporated herein by reference), Cholesterol (Sigma-Aldrich), andPEG-Ceramide C16 (Avanti Polar Lipids) can be used to preparelipid-dsRNA nanoparticles (i.e., LNP01 particles). Stock solutions ofeach in ethanol can be prepared as follows: ND98, 133 mg/ml;Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98,Cholesterol, and PEG-Ceramide C16 stock solutions can then be combinedin a, e.g., 42:48:10 molar ratio. The combined lipid solution can bemixed with aqueous dsRNA (e.g., in sodium acetate pH 5) such that thefinal ethanol concentration is about 35-45% and the final sodium acetateconcentration is about 100-300 mM. Lipid-dsRNA nanoparticles typicallyform spontaneously upon mixing. Depending on the desired particle sizedistribution, the resultant nanoparticle mixture can be extruded througha polycarbonate membrane (e.g., 100 nm cut-off) using, for example, athermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). Insome cases, the extrusion step can be omitted. Ethanol removal andsimultaneous buffer exchange can be accomplished by, for example,dialysis or tangential flow filtration. Buffer can be exchanged with,for example, phosphate buffered saline (PBS) at about pH 7, e.g., aboutpH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or aboutpH 7.4.

LNP01 formulations are described, e.g., in International ApplicationPublication No. WO 2008/042973, which is hereby incorporated byreference.

Additional exemplary lipid-dsRNA formulations are described in Table 1.

TABLE A cationic lipid/non-cationic lipid/cholesterol/PEG-lipidIonizable/Cationic Lipid conjugate Lipid:siRNA ratio SNALP-11,2-Dilinolenyloxy-N,N-dimethylaminopropaneDLinDMA/DPPC/Cholesterol/PEG- (DLinDMA) cDMA (57.1/7.1/34.4/1.4)lipid:siRNA ~7:1 2-XTC 2,2-Dilinoleyl-4-dimethylaminoethyl-XTC/DPPC/Cholesterol/PEG-cDMA [1,3]-dioxolane (XTC) 57.1/7.1/34.4/1.4lipid:siRNA ~7:1 LNP05 2,2-Dilinoleyl-4-dimethylaminoethyl-XTC/DSPC/Cholesterol/PEG-DMG [1,3]-dioxolane (XTC) 57.5/7.5/31.5/3.5lipid:siRNA ~6:1 LNP06 2,2-Dilinoleyl-4-dimethylaminoethyl-XTC/DSPC/Cholesterol/PEG-DMG [1,3]-dioxolane (XTC) 57.5/7.5/31.5/3.5lipid:siRNA ~11:1 LNP07 2,2-Dilinoleyl-4-dimethylaminoethyl-XTC/DSPC/Cholesterol/PEG-DMG [1,3]-dioxolane (XTC) 60/7.5/31/1.5,lipid:siRNA ~6:1 LNP08 2,2-Dilinoleyl-4-dimethylaminoethyl-XTC/DSPC/Cholesterol/PEG-DMG [1,3]-dioxolane (XTC) 60/7.5/31/1.5,lipid:siRNA ~11:1 LNP09 2,2-Dilinoleyl-4-dimethylaminoethyl-XTC/DSPC/Cholesterol/PEG-DMG [1,3]-dioxolane (XTC) 50/10/38.5/1.5Lipid:siRNA 10:1 LNP10 (3aR,5s,6aS)-N,N-dimethyl-2,2-ALN100/DSPC/Cholesterol/PEG-DMG di((9Z,12Z)-octadeca-9,12-dienyl)50/10/38.5/1.5 tetrahydro-3aH-cyclopenta[d][1,3]dioxol- Lipid:siRNA 10:15-amine (ALN100) LNP11 (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-MC-3/DSPC/Cholesterol/PEG-DMG tetraen-19-yl 4-(dimethylamino)butanoate50/10/38.5/1.5 (MC3) Lipid:siRNA 10:1 LNP12 1,1′-(2-(4-(2-((2-(bis(2-Tech G1/DSPC/Cholesterol/PEG-DMG hydroxydodecyl)amino)ethyl)(2-50/10/38.5/1.5 hydroxydodecyl)amino)ethyl)piperazin-1- Lipid:siRNA 10:1yl)ethylazanediyl)didodecan-2-ol (Tech G1) LNP13 XTCXTC/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 33:1 LNP14 MC3MC3/DSPC/Chol/PEG-DMG 40/15/40/5 Lipid:siRNA: 11:1 LNP15 MC3MC3/DSPC/Chol/PEG-DSG/GalNAc- PEG-DSG 50/10/35/4.5/0.5 Lipid:siRNA: 11:1LNP16 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP17MC3 MC3/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP18 MC3MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 12:1 LNP19 MC3MC3/DSPC/Chol/PEG-DMG 50/10/35/5 Lipid:siRNA: 8:1 LNP20 MC3MC3/DSPC/Chol/PEG-DPG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP21 C12-200C12-200/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP22 XTCXTC/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1

DSPC: distearoylphosphatidylcholine

DPPC: dipalmitoylphosphatidylcholine

PEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avgmol wt of 2000)

PEG-DSG: PEG-distyryl glycerol (C18-PEG, or PEG-C18) (PEG with avg molwt of 2000)

PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avg molwt of 2000)

SNALP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprisingformulations are described in International Publication No.WO2009/127060, filed Apr. 15, 2009, which is hereby incorporated byreference.

XTC comprising formulations are described in PCT Publication No. WO2010/088537, the entire contents of which are hereby incorporated hereinby reference.

MC3 comprising formulations are described, e.g., in U.S. Publication No.2010/0324120, filed Jun. 10, 2010, the entire contents of which arehereby incorporated by reference.

ALNY-100 comprising formulations are described in PCT Publication No. WO2010/054406, the entire contents of which are hereby incorporated hereinby reference.

C12-200 comprising formulations are described in PCT Publication No. WO2010/129709, the entire contents of which are hereby incorporated hereinby reference.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders can be desirable. In some embodiments, oralformulations are those in which dsRNAs featured in the invention areadministered in conjunction with one or more penetration enhancersurfactants and chelators. Suitable surfactants include fatty acidsand/or esters or salts thereof, bile acids and/or salts thereof.Suitable bile acids/salts include chenodeoxycholic acid (CDCA) andursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid,deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid,taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitablefatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g., sodium). In some embodiments, combinations of penetrationenhancers are used, for example, fatty acids/salts in combination withbile acids/salts. One exemplary combination is the sodium salt of lauricacid, capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAsfeatured in the invention can be delivered orally, in granular formincluding sprayed dried particles, or complexed to form micro ornanoparticles. DsRNA complexing agents include polyamino acids;polyimines; polyacrylates; polyalkylacrylates, polyoxethanes,polyalkylcyanoacrylates; cationized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses and starches. Suitable complexing agents include chitosan,N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine,polyspermines, protamine, polyvinylpyridine,polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g.,p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor dsRNAs and their preparation are described in detail in U.S. Pat.No. 6,887,906, US Publn. No. 20030027780, and U.S. Pat. No. 6,747,014,each of which is incorporated herein by reference.

Compositions and formulations for parenteral, intraparenchymal (into thebrain), intrathecal, intraventricular or intrahepatic administration caninclude sterile aqueous solutions which can also contain buffers,diluents and other suitable additives such as, but not limited to,penetration enhancers, carrier compounds and other pharmaceuticallyacceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions can be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids. Particularlypreferred are formulations that target the liver when treating hepaticdisorders such as hepatic carcinoma.

The pharmaceutical formulations of the present invention, which canconveniently be presented in unit dosage form, can be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention can be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention can also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions can further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension can also contain stabilizers.

C. Additional Formulations

i. Emulsions

The compositions of the present invention can be prepared and formulatedas emulsions. Emulsions are typically heterogeneous systems of oneliquid dispersed in another in the form of droplets usually exceeding0.1 μm in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms andDrug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004,Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al.,in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,Pa., 1985, p. 301). Emulsions are often biphasic systems comprising twoimmiscible liquid phases intimately mixed and dispersed with each other.In general, emulsions can be of either the water-in-oil (w/o) or theoil-in-water (o/w) variety. When an aqueous phase is finely divided intoand dispersed as minute droplets into a bulk oily phase, the resultingcomposition is called a water-in-oil (w/o) emulsion. Alternatively, whenan oily phase is finely divided into and dispersed as minute dropletsinto a bulk aqueous phase, the resulting composition is called anoil-in-water (o/w) emulsion. Emulsions can contain additional componentsin addition to the dispersed phases, and the active drug which can bepresent as a solution in either the aqueous phase, oily phase or itselfas a separate phase. Pharmaceutical excipients such as emulsifiers,stabilizers, dyes, and anti-oxidants can also be present in emulsions asneeded. Pharmaceutical emulsions can also be multiple emulsions that arecomprised of more than two phases such as, for example, in the case ofoil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.Such complex formulations often provide certain advantages that simplebinary emulsions do not. Multiple emulsions in which individual oildroplets of an o/w emulsion enclose small water droplets constitute aw/o/w emulsion. Likewise a system of oil droplets enclosed in globulesof water stabilized in an oily continuous phase provides an o/w/oemulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion can be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatcan be incorporated into either phase of the emulsion. Emulsifiers canbroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug DeliverySystems, Allen, L V., Popovich N G., and Ansel H C., 2004, LippincottWilliams & Wilkins (8th ed.), New York, N.Y.; Idson, in PharmaceuticalDosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (see e.g., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199).Surfactants are typically amphiphilic and comprise a hydrophilic and ahydrophobic portion. The ratio of the hydrophilic to the hydrophobicnature of the surfactant has been termed the hydrophile/lipophilebalance (HLB) and is a valuable tool in categorizing and selectingsurfactants in the preparation of formulations. Surfactants can beclassified into different classes based on the nature of the hydrophilicgroup: nonionic, anionic, cationic and amphoteric (see e.g., Ansel'sPharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V.,Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8thed.), New York, N.Y. Rieger, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that can readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used can be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture have been reviewedin the literature (see e.g., Ansel's Pharmaceutical Dosage Forms andDrug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004,Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsionformulations for oral delivery have been very widely used because ofease of formulation, as well as efficacy from an absorption andbioavailability standpoint (see e.g., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritivepreparations are among the materials that have commonly beenadministered orally as o/w emulsions.

ii. Microemulsions

In one embodiment of the present invention, the compositions of iRNAsand nucleic acids are formulated as microemulsions. A microemulsion canbe defined as a system of water, oil and amphiphile which is a singleoptically isotropic and thermodynamically stable liquid solution (seee.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems,Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams &Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical DosageForms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc.,New York, N.Y., volume 1, p. 245). Typically microemulsions are systemsthat are prepared by first dispersing an oil in an aqueous surfactantsolution and then adding a sufficient amount of a fourth component,generally an intermediate chain-length alcohol to form a transparentsystem. Therefore, microemulsions have also been described asthermodynamically stable, isotropically clear dispersions of twoimmiscible liquids that are stabilized by interfacial films ofsurface-active molecules (Leung and Shah, in: Controlled Release ofDrugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCHPublishers, New York, pages 185-215). Microemulsions commonly areprepared via a combination of three to five components that include oil,water, surfactant, cosurfactant and electrolyte. Whether themicroemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) typeis dependent on the properties of the oil and surfactant used and on thestructure and geometric packing of the polar heads and hydrocarbon tailsof the surfactant molecules (Schott, in Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (see e.g.,Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins(8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 335). Compared to conventional emulsions,microemulsions offer the advantage of solubilizing water-insoluble drugsin a formulation of thermodynamically stable droplets that are formedspontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions can, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase can typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase can include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (see e.g., U.S. Pat. Nos.6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (see e.g., U.S.Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides etal., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci.,1996, 85, 138-143). Often microemulsions can form spontaneously whentheir components are brought together at ambient temperature. This canbe particularly advantageous when formulating thermolabile drugs,peptides or iRNAs. Microemulsions have also been effective in thetransdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of iRNAs and nucleic acids from thegastrointestinal tract, as well as improve the local cellular uptake ofiRNAs and nucleic acids.

Microemulsions of the present invention can also contain additionalcomponents and additives such as sorbitan monostearate (Grill 3),Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the iRNAs and nucleic acidsof the present invention. Penetration enhancers used in themicroemulsions of the present invention can be classified as belongingto one of five broad categories—surfactants, fatty acids, bile salts,chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

iii. Microparticles

An iRNA agent of the invention may be incorporated into a particle,e.g., a microparticle. Microparticles can be produced by spray-drying,but may also be produced by other methods including lyophilization,evaporation, fluid bed drying, vacuum drying, or a combination of thesetechniques.

iv. Penetration Enhancers

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly iRNAs, to the skin of animals. Most drugs are present insolution in both ionized and nonionized forms. However, usually onlylipid soluble or lipophilic drugs readily cross cell membranes. It hasbeen discovered that even non-lipophilic drugs can cross cell membranesif the membrane to be crossed is treated with a penetration enhancer. Inaddition to aiding the diffusion of non-lipophilic drugs across cellmembranes, penetration enhancers also enhance the permeability oflipophilic drugs.

Penetration enhancers can be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants (see e.g., Malmsten, M.Surfactants and polymers in drug delivery, Informa Health Care, NewYork, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic DrugCarrier Systems, 1991, p. 92). Each of the above mentioned classes ofpenetration enhancers are described below in greater detail.

Surfactants (or “surface-active agents”) are chemical entities which,when dissolved in an aqueous solution, reduce the surface tension of thesolution or the interfacial tension between the aqueous solution andanother liquid, with the result that absorption of iRNAs through themucosa is enhanced. In addition to bile salts and fatty acids, thesepenetration enhancers include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (seee.g., Malmsten, M. Surfactants and polymers in drug delivery, InformaHealth Care, New York, N.Y., 2002; Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemicalemulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988,40, 252).

Various fatty acids and their derivatives which act as penetrationenhancers include, for example, oleic acid, lauric acid, capric acid(n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleicacid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C1-20 alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (see e.g.,Touitou, E., et al. Enhancement in Drug Delivery, CRC Press, Danvers,Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug CarrierSystems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic DrugCarrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol.,1992, 44, 651-654).

The physiological role of bile includes the facilitation of dispersionand absorption of lipids and fat-soluble vitamins (see e.g., Malmsten,M. Surfactants and polymers in drug delivery, Informa Health Care, NewYork, N.Y., 2002; Brunton, Chapter 38 in: Goodman & Gilman's ThePharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds.,McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts,and their synthetic derivatives, act as penetration enhancers. Thus theterm “bile salts” includes any of the naturally occurring components ofbile as well as any of their synthetic derivatives. Suitable bile saltsinclude, for example, cholic acid (or its pharmaceutically acceptablesodium salt, sodium cholate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodiumglycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (see e.g.,Malmsten, M. Surfactants and polymers in drug delivery, Informa HealthCare, New York, N.Y., 2002; Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In:Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., MackPublishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto etal., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm.Sci., 1990, 79, 579-583).

Chelating agents, as used in connection with the present invention, canbe defined as compounds that remove metallic ions from solution byforming complexes therewith, with the result that absorption of iRNAsthrough the mucosa is enhanced. With regards to their use as penetrationenhancers in the present invention, chelating agents have the addedadvantage of also serving as DNase inhibitors, as most characterized DNAnucleases require a divalent metal ion for catalysis and are thusinhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618,315-339). Suitable chelating agents include but are not limited todisodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates(e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acylderivatives of collagen, laureth-9 and N-amino acyl derivatives ofbeta-diketones (enamines)(see e.g., Katdare, A. et al., Excipientdevelopment for pharmaceutical, biotechnology, and drug delivery, CRCPress, Danvers, Mass., 2006; Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. ControlRel., 1990, 14, 43-51).

As used herein, non-chelating non-surfactant penetration enhancingcompounds can be defined as compounds that demonstrate insignificantactivity as chelating agents or as surfactants but that nonethelessenhance absorption of iRNAs through the alimentary mucosa (see e.g.,Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33). This class of penetration enhancers includes, for example,unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanonederivatives (Lee et al., Critical Reviews in Therapeutic Drug CarrierSystems, 1991, page 92); and non-steroidal anti-inflammatory agents suchas diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al.,J. Pharm. Pharmacol., 1987, 39, 621-626).

Agents that enhance uptake of iRNAs at the cellular level can also beadded to the pharmaceutical and other compositions of the presentinvention. For example, cationic lipids, such as lipofectin (Junichi etal, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, andpolycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof dsRNAs. Examples of commercially available transfection reagentsinclude, for example Lipofectamine™ (Invitrogen; Carlsbad, Calif.),Lipofectamine 2000™ (Invitrogen; Carlsbad, Calif.), 293Fectin™(Invitrogen; Carlsbad, Calif.), Cellfectin™ (Invitrogen; Carlsbad,Calif.), DMRIE-C™ (Invitrogen; Carlsbad, Calif.), FreeStyle™ MAX(Invitrogen; Carlsbad, Calif.), Lipofectamine™ 2000 CD (Invitrogen;Carlsbad, Calif.), Lipofectamine™ (Invitrogen; Carlsbad, Calif.),iRNAMAX (Invitrogen; Carlsbad, Calif.), Oligofectamine™ (Invitrogen;Carlsbad, Calif.), Optifect™ (Invitrogen; Carlsbad, Calif.), X-tremeGENEQ2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAPLiposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPERLiposomal Transfection Reagent (Grenzacherstrasse, Switzerland), orFugene (Grenzacherstrasse, Switzerland), Transfectam® Reagent (Promega;Madison, Wis.), TransFast™ Transfection Reagent (Promega; Madison,Wis.), Tfx™-20 Reagent (Promega; Madison, Wis.), Tfx™-50 Reagent(Promega; Madison, Wis.), DreamFect™ (OZ Biosciences; Marseille,France), EcoTransfect (OZ Biosciences; Marseille, France), TransPass^(a)D1 Transfection Reagent (New England Biolabs; Ipswich, Mass., USA),LyoVec™/LipoGen™ (Invitrogen; San Diego, Calif., USA), PerFectinTransfection Reagent (Genlantis; San Diego, Calif., USA), NeuroPORTERTransfection Reagent (Genlantis; San Diego, Calif., USA), GenePORTERTransfection reagent (Genlantis; San Diego, Calif., USA), GenePORTER 2Transfection reagent (Genlantis; San Diego, Calif., USA), CytofectinTransfection Reagent (Genlantis; San Diego, Calif., USA), BaculoPORTERTransfection Reagent (Genlantis; San Diego, Calif., USA), TroganPORTER™transfection Reagent (Genlantis; San Diego, Calif., USA), RiboFect(Bioline; Taunton, Mass., USA), PlasFect (Bioline; Taunton, Mass., USA),UniFECTOR (B-Bridge International; Mountain View, Calif., USA),SureFECTOR (B-Bridge International; Mountain View, Calif., USA), orHiFect™ (B-Bridge International, Mountain View, Calif., USA), amongothers.

Other agents can be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

v. Carriers

Certain compositions of the present invention also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The coadministration of a nucleic acid and a carriercompound, typically with an excess of the latter substance, can resultin a substantial reduction of the amount of nucleic acid recovered inthe liver, kidney or other extracirculatory reservoirs, presumably dueto competition between the carrier compound and the nucleic acid for acommon receptor. For example, the recovery of a partiallyphosphorothioate dsRNA in hepatic tissue can be reduced when it iscoadministered with polyinosinic acid, dextran sulfate, polycytidic acidor 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao etal., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl.Acid Drug Dev., 1996, 6, 177-183.

vi. Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient can be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc).

Pharmaceutically acceptable organic or inorganic excipients suitable fornon-parenteral administration which do not deleteriously react withnucleic acids can also be used to formulate the compositions of thepresent invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids can includesterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions of the nucleic acids inliquid or solid oil bases. The solutions can also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

Suitable pharmaceutically acceptable excipients include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

vii. Other Components

The compositions of the present invention can additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions can contain additional, compatible, pharmaceutically-activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or can contain additionalmaterials useful in physically formulating various dosage forms of thecompositions of the present invention, such as dyes, flavoring agents,preservatives, antioxidants, opacifiers, thickening agents andstabilizers. However, such materials, when added, should not undulyinterfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Aqueous suspensions can contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension can also contain stabilizers.

In some embodiments, pharmaceutical compositions featured in theinvention include (a) one or more iRNA compounds and (b) one or moreagents which function by a non-iRNA mechanism and which are useful intreating a hemolytic disorder. Examples of such agents include, but arenot limited to an anti-inflammatory agent, anti-steatosis agent,anti-viral, and/or anti-fibrosis agent.

In addition, other substances commonly used to protect the liver, suchas silymarin, can also be used in conjunction with the iRNAs describedherein. Other agents useful for treating liver diseases includetelbivudine, entecavir, and protease inhibitors such as telaprevir andother disclosed, for example, in Tung et al., U.S. ApplicationPublication Nos. 2005/0148548, 2004/0167116, and 2003/0144217; and inHale et al., U.S. Application Publication No. 2004/0127488.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofcompositions featured herein in the invention lies generally within arange of circulating concentrations that include the ED50 with little orno toxicity. The dosage can vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the methods featured in the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose can be formulated in animal models to achieve acirculating plasma concentration range of the compound or, whenappropriate, of the polypeptide product of a target sequence (e.g.,achieving a decreased concentration of the polypeptide) that includesthe IC50 (i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma can be measured, for example, by highperformance liquid chromatography.

In addition to their administration, as discussed above, the iRNAsfeatured in the invention can be administered in combination with otherknown agents effective in treatment of pathological processes mediatedby TTR expression. In any event, the administering physician can adjustthe amount and timing of iRNA administration on the basis of resultsobserved using standard measures of efficacy known in the art ordescribed herein.

VII. Methods for Inhibiting TTR Expression

The present invention also provides methods of inhibiting expression ofa transthyretin (TTR) in a cell. The methods include contacting a cellwith an RNAi agent, e.g., double stranded RNAi agent, in an amounteffective to inhibit expression of TTR in the cell, thereby inhibitingexpression of TTR in the cell.

Contacting of a cell with an RNAi agent, e.g., a double stranded RNAiagent, may be done in vitro or in vivo. Contacting a cell in vivo withthe RNAi agent includes contacting a cell or group of cells within asubject, e.g., a human subject, with the RNAi agent. Combinations of invitro and in vivo methods of contacting a cell or group of cells arealso possible. Contacting a cell or a group of cells may be direct orindirect, as discussed above. Furthermore, contacting a cell or a groupof cells may be accomplished via a targeting ligand, including anyligand described herein or known in the art. In preferred embodiments,the targeting ligand is a carbohydrate moiety, e.g., a GalNAc₃ ligand,or any other ligand that directs the RNAi agent to a site of interest,e.g., the liver of a subject.

The term “inhibiting,” as used herein, is used interchangeably with“reducing,” “silencing,” “downregulating”, “suppressing”, and othersimilar terms, and includes any level of inhibition. Preferablyinhibiting includes a statistically significant or clinicallysignificant inhibition.

The phrase “inhibiting expression of a TTR” is intended to refer toinhibition of expression of any TTR gene (such as, e.g., a mouse TTRgene, a rat TTR gene, a monkey TTR gene, or a human TTR gene) as well asvariants or mutants of a TTR gene. Thus, the TTR gene may be a wild-typeTTR gene, a mutant TTR gene (such as a mutant TTR gene giving rise toamyloid deposition), or a transgenic TTR gene in the context of agenetically manipulated cell, group of cells, or organism.

“Inhibiting expression of a TTR gene” includes any level of inhibitionof a TTR gene, e.g., at least partial suppression of the expression of aTTR gene. The expression of the TTR gene may be assessed based on thelevel, or the change in the level, of any variable associated with TTRgene expression, e.g., TTR mRNA level, TTR protein level, or the numberor extent of amyloid deposits. This level may be assessed in anindividual cell or in a group of cells, including, for example, a samplederived from a subject.

Inhibition may be assessed by a decrease in an absolute or relativelevel of one or more variables that are associated with TTR expressioncompared with a control level. The control level may be any type ofcontrol level that is utilized in the art, e.g., a pre-dose baselinelevel, or a level determined from a similar subject, cell, or samplethat is untreated or treated with a control (such as, e.g., buffer onlycontrol or inactive agent control).

In some embodiments of the methods of the invention, expression of a TTRgene is inhibited by at least about 5%, at least about 10%, at leastabout 15%, at least about 20%, at least about 25%, at least about 30%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 91%, at least about 92%,at least about 93%, at least about 94%. at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, at least about 99%%,or to below the level of detection of the assay. In some embodiments,the inhibition of expression of a TTRgene results in normalization ofthe level of the TTR gene such that the difference between the levelbefore treatment and a normal control level is reduced by at least 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. Insome embodiments, the inhibition is a clinically relevant inhibition.

Inhibition of the expression of a TTR gene may be manifested by areduction of the amount of mRNA expressed by a first cell or group ofcells (such cells may be present, for example, in a sample derived froma subject) in which a TTR gene is transcribed and which has or have beentreated (e.g., by contacting the cell or cells with an RNAi agent of theinvention, or by administering an RNAi agent of the invention to asubject in which the cells are or were present) such that the expressionof a TTR gene is inhibited, as compared to a second cell or group ofcells substantially identical to the first cell or group of cells butwhich has not or have not been so treated (control cell(s)). Inpreferred embodiments, the inhibition is assessed by expressing thelevel of mRNA in treated cells as a percentage of the level of mRNA incontrol cells, using the following formula:

${\frac{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right) - \left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{treated}\mspace{14mu}{cells}} \right)}{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right)} \cdot 100}\%$

Alternatively, inhibition of the expression of a TTR gene may beassessed in terms of a reduction of a parameter that is functionallylinked to TTR gene expression, e.g., TTR protein expression, retinolbinding protein level, vitamin A level, or presence of amyloid depositscomprising TTR. TTR gene silencing may be determined in any cellexpressing TTR, either constitutively or by genomic engineering, and byany assay known in the art. The liver is the major site of TTRexpression. Other significant sites of expression include the choroidplexus, retina and pancreas.

Inhibition of the expression of a TTR protein may be manifested by areduction in the level of the TTR protein that is expressed by a cell orgroup of cells (e.g., the level of protein expressed in a sample derivedfrom a subject). As explained above for the assessment of mRNAsuppression, the inhibition of protein expression levels in a treatedcell or group of cells may similarly be expressed as a percentage of thelevel of protein in a control cell or group of cells.

A control cell or group of cells that may be used to assess theinhibition of the expression of a TTR gene includes a cell or group ofcells that has not yet been contacted with an RNAi agent of theinvention. For example, the control cell or group of cells may bederived from an individual subject (e.g., a human or animal subject)prior to treatment of the subject with an RNAi agent.

The level of TTR mRNA that is expressed by a cell or group of cells, orthe level of circulating TTR mRNA, may be determined using any methodknown in the art for assessing mRNA expression. In one embodiment, thelevel of expression of TTR in a sample is determined by detecting atranscribed polynucleotide, or portion thereof, e.g., mRNA of the TTRgene. RNA may be extracted from cells using RNA extraction techniquesincluding, for example, using acid phenol/guanidine isothiocyanateextraction (RNAzol B; Biogenesis), RNeasy RNA preparation kits (Qiagen)or PAXgene (PreAnalytix, Switzerland). Typical assay formats utilizingribonucleic acid hybridization include nuclear run-on assays, RT-PCR,RNase protection assays (Melton et al., Nuc. Acids Res. 12:7035),Northern blotting, in situ hybridization, and microarray analysis.Circulating TTR mRNA may be detected using methods the described inPCT/US2012/043584, the entire contents of which are hereby incorporatedherein by reference.

In one embodiment, the level of expression of TTR is determined using anucleic acid probe. The term “probe”, as used herein, refers to anymolecule that is capable of selectively binding to a specific TTR.Probes can be synthesized by one of skill in the art, or derived fromappropriate biological preparations. Probes may be specifically designedto be labeled. Examples of molecules that can be utilized as probesinclude, but are not limited to, RNA, DNA, proteins, antibodies, andorganic molecules.

Isolated mRNA can be used in hybridization or amplification assays thatinclude, but are not limited to, Southern or Northern analyses,polymerase chain reaction (PCR) analyses and probe arrays. One methodfor the determination of mRNA levels involves contacting the isolatedmRNA with a nucleic acid molecule (probe) that can hybridize to TTRmRNA. In one embodiment, the mRNA is immobilized on a solid surface andcontacted with a probe, for example by running the isolated mRNA on anagarose gel and transferring the mRNA from the gel to a membrane, suchas nitrocellulose. In an alternative embodiment, the probe(s) areimmobilized on a solid surface and the mRNA is contacted with theprobe(s), for example, in an Affymetrix gene chip array. A skilledartisan can readily adapt known mRNA detection methods for use indetermining the level of TTR mRNA.

An alternative method for determining the level of expression of TTR ina sample involves the process of nucleic acid amplification and/orreverse transcriptase (to prepare cDNA) of for example mRNA in thesample, e.g., by RT-PCR (the experimental embodiment set forth inMullis, 1987, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany(1991) Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequencereplication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh et al. (1989)Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi etal. (1988) Bio/Technology 6:1197), rolling circle replication (Lizardiet al., U.S. Pat. No. 5,854,033) or any other nucleic acid amplificationmethod, followed by the detection of the amplified molecules usingtechniques well known to those of skill in the art. These detectionschemes are especially useful for the detection of nucleic acidmolecules if such molecules are present in very low numbers. Inparticular aspects of the invention, the level of expression of TTR isdetermined by quantitative fluorogenic RT-PCR (i.e., the TaqMan™System).

The expression levels of TTR mRNA may be monitored using a membrane blot(such as used in hybridization analysis such as Northern, Southern, dot,and the like), or microwells, sample tubes, gels, beads or fibers (orany solid support comprising bound nucleic acids). See U.S. Pat. Nos.5,770,722, 5,874,219, 5,744,305, 5,677,195 and 5,445,934, which areincorporated herein by reference. The determination of TTR expressionlevel may also comprise using nucleic acid probes in solution.

In preferred embodiments, the level of mRNA expression is assessed usingbranched DNA (bDNA) assays or real time PCR (qPCR). The use of thesemethods is described and exemplified in the Examples presented herein.

The level of TTR protein expression may be determined using any methodknown in the art for the measurement of protein levels. Such methodsinclude, for example, electrophoresis, capillary electrophoresis, highperformance liquid chromatography (HPLC), thin layer chromatography(TLC), hyperdiffusion chromatography, fluid or gel precipitin reactions,absorption spectroscopy, a colorimetric assays, spectrophotometricassays, flow cytometry, immunodiffusion (single or double),immunoelectrophoresis, Western blotting, radioimmunoassay (RIA),enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays,electrochemiluminescence assays, and the like.

In some embodiments, the efficacy of the methods of the invention can bemonitored by detecting or monitoring a reduction in an amyloid TTRdeposit. Reducing an amyloid TTR deposit, as used herein, includes anydecrease in the size, number, or severity of TTR deposits, or to aprevention or reduction in the formation of TTR deposits, within anorgan or area of a subject, as may be assessed in vitro or in vivo usingany method known in the art. For example, some methods of assessingamyloid deposits are described in Gertz, M. A. & Rajukumar, S. V.(Editors) (2010), Amyloidosis: Diagnosis and Treatment, New York: HumanaPress. Methods of assessing amyloid deposits may include biochemicalanalyses, as well as visual or computerized assessment of amyloiddeposits, as made visible, e.g., using immunohistochemical staining,fluorescent labeling, light microscopy, electron microscopy,fluorescence microscopy, or other types of microscopy. Invasive ornoninvasive imaging modalities, including, e.g., CT, PET, or NMR/MRIimaging may be employed to assess amyloid deposits.

The methods of the invention may reduce TTR deposits in any number oftissues or regions of the body including but not limited to the heart,liver, spleen, esophagus, stomach, intestine (ileum, duodenum andcolon), brain, sciatic nerve, dorsal root ganglion, kidney and retina.

The term “sample” as used herein refers to a collection of similarfluids, cells, or tissues isolated from a subject, as well as fluids,cells, or tissues present within a subject. Examples of biologicalfluids include blood, serum and serosal fluids, plasma, lymph, urine,cerebrospinal fluid, saliva, ocular fluids, and the like. Tissue samplesmay include samples from tissues, organs or localized regions. Forexample, samples may be derived from particular organs, parts of organs,or fluids or cells within those organis. In certain embodiments, samplesmay be derived from the liver (e.g., whole liver or certain segments ofliver or certain types of cells in the liver, such as, e.g.,hepatocytes), the retina or parts of the retina (e.g., retinal pigmentepithelium), the central nervous system or parts of the central nervoussystem (e.g., ventricles or choroid plexus), or the pancreas or certaincells or parts of the pancreas. In preferred embodiments, a “samplederived from a subject” refers to blood drawn from the subject or plasmaderived therefrom. In further embodiments, a “sample derived from asubject” refers to liver tissue or retinal tissue derived from thesubject.

In some embodiments of the methods of the invention, the RNAi agent isadministered to a subject such that the RNAi agent is delivered to aspecific site within the subject. The inhibition of expression of TTRmay be assessed using measurements of the level or change in the levelof TTR mRNA or TTR protein in a sample derived from fluid or tissue fromthe specific site within the subject. In preferred embodiments, the siteis selected from the group consisting of liver, choroid plexus, retina,and pancreas. The site may also be a subsection or subgroup of cellsfrom any one of the aforementioned sites (e.g., hepatocytes or retinalpigment epithelium). The site may also include cells that express aparticular type of receptor (e.g., hepatocytes that express theasialogycloprotein receptor).

VIII. Methods for Treating or Preventing a TTR-Associated Disease

The present invention also provides methods for treating or preventing aTTR-associated disease in a subject. The methods include administeringto the subject a therapeutically effective amount or prophylacticallyeffective amount of an RNAi agent of the invention.

As used herein, a “subject” is an animal, such as a mammal, including aprimate (such as a human, a non-human primate, e.g., a monkey, and achimpanzee), a non-primate (such as a cow, a pig, a camel, a llama, ahorse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog,a rat, a mouse, a horse, and a whale), or a bird (e.g., a duck or agoose). A subject may include a transgenic organism.

In an embodiment, the subject is a human, such as a human being treatedor assessed for a disease, disorder or condition that would benefit fromreduction in TTR gene expression; a human at risk for a disease,disorder or condition that would benefit from reduction in TTR geneexpression; a human having a disease, disorder or condition that wouldbenefit from reduction in TTR gene expression; and/or human beingtreated for a disease, disorder or condition that would benefit fromreduction in TTR gene expression, as described herein.

In some embodiments, the subject is suffering from a TTR-associateddisease. In other embodiments, the subject is a subject at risk fordeveloping a TTR-associated disease, e.g., a subject with a TTR genemutation that is associated with the development of a TTR associateddisease (e.g., before the onset of signs or symptoms suggesting thedevelopment of TTR amyloidosis), a subject with a family history ofTTR-associated disease (e.g., before the onset of signs or symptomssuggesting the development of TTR amyloidosis), or a subject who hassigns or symptoms suggesting the development of TTR amyloidosis.

A “TTR-associated disease,” as used herein, includes any disease causedby or associated with the formation of amyloid deposits in which thefibril precursors consist of variant or wild-type TTR protein. Mutantand wild-type TTR give rise to various forms of amyloid deposition(amyloidosis). Amyloidosis involves the formation and aggregation ofmisfolded proteins, resulting in extracellular deposits that impairorgan function. Clinical syndromes associated with TTR aggregationinclude, for example, senile systemic amyloidosis (SSA); systemicfamilial amyloidosis; familial amyloidotic polyneuropathy (FAP);familial amyloidotic cardiomyopathy (FAC); and leptomeningealamyloidosis, also known as leptomeningeal or meningocerebrovascularamyloidosis, central nervous system (CNS) amyloidosis, or amyloidosisVII form.

In one embodiment, the RNAi agents of the invention are administered tosubjects suffering from familial amyloidotic cardiomyopathy (FAC). Inanother embodiment, the RNAi agents of the invention are administered tosubjects suffering from FAC with a mixed phenotype, i.e., a subjecthaving both cardiac and neurological impairments. In yet anotherembodiment, the RNAi agents of the invention are administered tosubjects suffering from FAP with a mixed phenotype, i.e., a subjecthaving both neurological and cardiac impairments. In one embodiment, theRNAi agents of the invention are administered to subjects suffering fromFAP that has been treated with an orthotopic liver transplantation(OLT). In another embodiment, the RNAi agents of the invention areadministered to subjects suffering from senile systemic amyloidosis(SSA). In other embodiments of the methods of the invention, RNAi agentsof the invention are administered to subjects suffering from familialamyloidotic cardiomyopathy (FAC) and senile systemic amyloidosis (SSA).Normal-sequence TTR causes cardiac amyloidosis in people who are elderlyand is termed senile systemic amyloidosis (SSA) (also called senilecardiac amyloidosis (SCA) or cardiac amyloidosis). SSA often isaccompanied by microscopic deposits in many other organs. TTR mutationsaccelerate the process of TTR amyloid formation and are the mostimportant risk factor for the development of clinically significant TTRamyloidosis (also called ATTR (amyloidosis-transthyretin type)). Morethan 85 amyloidogenic TTR variants are known to cause systemic familialamyloidosis.

In some embodiments of the methods of the invention, RNAi agents of theinvention are administered to subjects suffering from transthyretin(TTR)-related familial amyloidotic polyneuropathy (FAP). Such subjectsmay suffer from ocular manifestations, such as vitreous opacity andglaucoma. It is known to one of skill in the art that amyloidogenictransthyretin (ATTR) synthesized by retinal pigment epithelium (RPE)plays important roles in the progression of ocular amyloidosis. Previousstudies have shown that panretinal laser photocoagulation, which reducedthe RPE cells, prevented the progression of amyloid deposition in thevitreous, indicating that the effective suppression of ATTR expressionin RPE may become a novel therapy for ocular amyloidosis (see, e.g.,Kawaji, T., et al., Ophthalmology. (2010) 117: 552-555). The methods ofthe invention are useful for treatment of ocular manifestations of TTRrelated FAP, e.g., ocular amyloidosis. The RNAi agent can be deliveredin a manner suitable for targeting a particular tissue, such as the eye.Modes of ocular delivery include retrobulbar, subcutaneous eyelid,subconjunctival, subtenon, anterior chamber or intravitreous injection(or internal injection or infusion). Specific formulations for oculardelivery include eye drops or ointments.

Another TTR-associated disease is hyperthyroxinemia, also known as“dystransthyretinemic hyperthyroxinemia” or “dysprealbuminemichyperthyroxinemia”. This type of hyperthyroxinemia may be secondary toan increased association of thyroxine with TTR due to a mutant TTRmolecule with increased affinity for thyroxine. See, e.g., Moses et al.(1982) J. Clin. Invest., 86, 2025-2033.

The RNAi agents of the invention may be administered to a subject usingany mode of administration known in the art, including, but not limitedto subcutaneous, intravenous, intramuscular, intraocular,intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic,cerebrospinal, and any combinations thereof.

In preferred embodiments, the agents are administered to the subjectsubcutaneously. In some embodiments, a subject is administered a singledose of an RNAi agent via subcutaneous injection, e.g., abdominal,thigh, or upper arm injection. In other embodiments, a subject isadministered a split dose of an RNAi agent via subcutaneous injection.In one embodiment, the split dose of the RNAi agent is administered tothe subject via subcutaneous injection at two different anatomicallocations on the subject. For example, the subject may be subcutaneouslyinjected with a split dose of about 250 mg (e.g., about half of a 500 mgdose) in the right arm and about 250 mg in the left arm. In someembodiments of the invention, the subcutaneous administration isself-administration via, e.g., a pre-filled syringe or auto-injectorsyringe. In some embodiments, a dose of the RNAi agent for subcutaneousadministration is contained in a volume of less than or equal to one mlof, e.g., a pharmaceutically acceptable carrier.

In some embodiments, the administration is via a depot injection. Adepot injection may release the RNAi agent in a consistent way over aprolonged time period. Thus, a depot injection may reduce the frequencyof dosing needed to obtain a desired effect, e.g., a desired inhibitionof TTR, or a therapeutic or prophylactic effect. A depot injection mayalso provide more consistent serum concentrations. Depot injections mayinclude subcutaneous injections or intramuscular injections. Inpreferred embodiments, the depot injection is a subcutaneous injection.

In some embodiments, the administration is via a pump. The pump may bean external pump or a surgically implanted pump. In certain embodiments,the pump is a subcutaneously implanted osmotic pump. In otherembodiments, the pump is an infusion pump. An infusion pump may be usedfor intravenous, subcutaneous, arterial, or epidural infusions. Inpreferred embodiments, the infusion pump is a subcutaneous infusionpump. In other embodiments, the pump is a surgically implanted pump thatdelivers the RNAi agent to the liver.

In embodiments in which the RNAi agent is administered via asubcutaneous infusion pump, a single dose of the RNAi agent may beadministered to the subject over a period of time of about 45 minutes toabout 5 minutes, e.g., about 45 minutes, about 40 minutes, about 35minutes, about 30 minutes, about 25 minutes, about 20 minutes, about 15minutes, about 10 minutes, or about 5 minutes.

Other modes of administration include epidural, intracerebral,intracerebroventricular, nasal administration, intraarterial,intracardiac, intraosseous infusion, intrathecal, and intravitreal, andpulmonary. The mode of administration may be chosen based upon whetherlocal or systemic treatment is desired and based upon the area to betreated. The route and site of administration may be chosen to enhancetargeting.

In some embodiments, the RNAi agent is administered to a subject in anamount effective to inhibit TTR expression in a cell within the subject.The amount effective to inhibit TTR expression in a cell within asubject may be assessed using methods discussed above, including methodsthat involve assessment of the inhibition of TTR mRNA, TTR protein, orrelated variables, such as amyloid deposits.

In some embodiments, the RNAi agent is administered to a subject in atherapeutically or prophylactically effective amount.

“Therapeutically effective amount,” as used herein, is intended toinclude the amount of an RNAi agent that, when administered to a patientfor treating a TTR associated disease, is sufficient to effect treatmentof the disease (e.g., by diminishing, ameliorating or maintaining theexisting disease or one or more symptoms of disease). The“therapeutically effective amount” may vary depending on the RNAi agent,how the agent is administered, the disease and its severity and thehistory, age, weight, family history, genetic makeup, stage ofpathological processes mediated by TTR expression, the types ofpreceding or concomitant treatments, if any, and other individualcharacteristics of the patient to be treated.

“Prophylactically effective amount,” as used herein, is intended toinclude the amount of an RNAi agent that, when administered to a subjectwho does not yet experience or display symptoms of a TTR-associateddisease, but who may be predisposed to the disease, is sufficient toprevent or ameliorate the disease or one or more symptoms of thedisease. Symptoms that may be ameliorated include sensory neuropathy(e.g., paresthesia, hypesthesia in distal limbs), autonomic neuropathy(e.g., gastrointestinal dysfunction, such as gastric ulcer, ororthostatic hypotension), motor neuropathy, seizures, dementia,myelopathy, polyneuropathy, carpal tunnel syndrome, autonomicinsufficiency, cardiomyopathy, vitreous opacities, renal insufficiency,nephropathy, substantially reduced mBMI (modified Body Mass Index),cranial nerve dysfunction, and corneal lattice dystrophy. Amelioratingthe disease includes slowing the course of the disease or reducing theseverity of later-developing disease. The “prophylactically effectiveamount” may vary depending on the RNAi agent, how the agent isadministered, the degree of risk of disease, and the history, age,weight, family history, genetic makeup, the types of preceding orconcomitant treatments, if any, and other individual characteristics ofthe patient to be treated.

A “therapeutically-effective amount” or “prophylacticaly effectiveamount” also includes an amount of an RNAi agent that produces somedesired local or systemic effect at a reasonable benefit/risk ratioapplicable to any treatment. RNAi agents employed in the methods of thepresent invention may be administered in a sufficient amount to producea reasonable benefit/risk ratio applicable to such treatment.

As used herein, the phrases “therapeutically effective amount” and“prophylactically effective amount” also include an amount that providesa benefit in the treatment, prevention, or management of pathologicalprocesses or symptom(s) of pathological processes mediated by TTRexpression. Symptoms of TTR amyloidosis include sensory neuropathy (e.g.paresthesia, hypesthesia in distal limbs), autonomic neuropathy (e.g.,gastrointestinal dysfunction, such as gastric ulcer, or orthostatichypotension), motor neuropathy, seizures, dementia, myelopathy,polyneuropathy, carpal tunnel syndrome, autonomic insufficiency,cardiomyopathy, vitreous opacities, renal insufficiency, nephropathy,substantially reduced mBMI (modified Body Mass Index), cranial nervedysfunction, and corneal lattice dystrophy.

In one embodiment, for example, when the subject has FAP, FAP with mixedphenotype, FAC with mixed phenotype, or FAP and has had an OLT,treatment of the subject with a dsRNA agent of the invention slows theprogression of neuropathy. In another embodiment, for example, when thesubject has FAP, FAP with mixed phenotype, FAC with mixed phenotype,SSA, or FAP and has had an OLT, treatment of the subject with a dsRNAagent of the invention slows the progression of neuropathy andcardiomyopathy.

For example, in one embodiment, the methods of the invention slow,reduce or arrest neurological impairment. Any suitable measure ofneurological impairment can be used to determine whether a subject hasreduced, slowed, or arrested neurological impairment.

One suitable measure is a Neuropathy Impairment Score (NIS). NIS refersto a scoring system that measures weakness, sensation, and reflexes,especially with respect to peripheral neuropathy. The NIS scoreevaluates a standard group of muscles for weakness (1 is 25% weak, 2 is50% weak, 3 is 75% weak, 3.25 is movement against gravity, 3.5 ismovement with gravity eliminated, 3.75 is muscle flicker withoutmovement, and 4 is paralyzed), a standard group of muscle stretchreflexes (0 is normal, 1 is decreased, 2 is absent), and touch-pressure,vibration, joint position and motion, and pinprick (all graded on indexfinger and big toe: 0 is normal, 1 is decreased, 2 is absent).Evaluations are corrected for age, gender, and physical fitness.

In one embodiment, the methods of the invention reduce a NIS by at least10%. In other embodiments, the methods of the invention result in areduction of NIS by at least 5, 10, 15, 20, 25, 30, 40, or by at least50%. In other embodiments, the methods arrest an increasing NIS score,e.g., the method results in a 0% increase of the NIS score. In yet otherembodiments, the methods of the invention slow the rate at which an NISscore increase, e.g., the rate of increase of an NIS score in a subjecttreated with an RNAi agent of the invention as compared to the rate ofincrease of an NIS score in a subject that is not treated with an RNAiagent of the invention.

Methods for determining an NIS in a human subject are well known to oneof skill in the art and can be found in, for example, Dyck, P J et al.,(1997) Neurology 1997. 49(1): pgs. 229-239); Dyck P J. (1988) MuscleNerve. January; 11(1):21-32.

Another suitable measurement of neurological impairment is a ModifiedNeuropathy Impairment Score (mNIS+7). As known to one of ordinary skillin the art, mNIS+7 refers to a clinical exam-based assessment ofneurologic impairment (NIS) combined with electrophysiologic measures ofsmall and large nerve fiber function (NCS and QST), and measurement ofautonomic function (postural blood pressure). The mNIS+7 score is amodification of the NIS+7 score (which represents NIS plus seven tests).NIS+7 analyzes weakness and muscle stretch reflexes. Five of the seventests include attributes of nerve conduction. These attributes are theperoneal nerve compound muscle action potential amplitude, motor nerveconduction velocity and motor nerve distal latency (MNDL), tibial MNDL,and sural sensory nerve action potential amplitudes. These values arecorrected for variables of age, gender, height, and weight. Theremaining two of the seven tests include vibratory detection thresholdand heart rate decrease with deep breathing.

The mNIS+7 score modifies NIS+7 to take into account the use of SmartSomatotopic Quantitative Sensation Testing, new autonomic assessments,and the use of compound muscle action potential of amplitudes of theulnar, peroneal, and tibial nerves, and sensory nerve action potentialsof the ulnar and sural nerves (Suanprasert, N. et al., (2014) J. Neurol.Sci., 344(1-2): pgs. 121-128).

In one embodiment, the methods of the invention reduce an mNIS+7 by atleast 10%. In other embodiments, the methods of the invention result ina reduction of an mNIS+7 score by at least 5, 10, 15, 20, 25, 30, 40, orby at least 50%. In other embodiments, the methods arrest an increasingmNIS+7, e.g., the method results in a 0% increase of the mNIS+7. In yetother embodiments, the methods of the invention slow the rate at whichan NIS+7 score increase, e.g., the rate of increase of an NIS+7 score ina subject treated with an RNAi agent of the invention as compared to therate of increase of an NIS+7 score in a subject that is not treated withan RNAi agent of the invention.

The therapeutic and prophylactic methods of the present invention mayalso improve other clinical parameters, such a walking ability, in thesubject being treated. For example, during or following a treatmentperiod a subject may have an increased exercise capacity or activity.

Any suitable measure of exercise capacity can be used to determinewhether a subject has an increased exercise capacity or activity. Onesuitable measure is a 6-minute walk test (6MWT), which measures how farthe subject can walk in 6 minutes, i.e., the 6-minute walk distance(6MWD). In one embodiment, the methods of the invention provide to thesubject an increase from baseline in the 6MWD by at least about 10minutes, e.g., about 10, 15, 20, or about 30 minutes.

A therapeutically effective amount and prophylactically effective amountof an RNAi agent of the invention also includes an amount that improvesone or more quality of life parameters versus baseline, for example anincrease in score on at least one of the SF-36® health survey functionalscales; and/or an increased longevity; and/or decreased hospitalization.

Any suitable measure quality of life may be used. For example, theSF-36® health survey provides a self-reporting, multi-item scalemeasuring eight health parameters: physical functioning, rolelimitations due to physical health problems, bodily pain, generalhealth, vitality (energy and fatigue), social functioning, rolelimitations due to emotional problems, and mental health (psychologicaldistress and psychological well-being). The survey also provides aphysical component summary and a mental component summary. In oneembodiment, the methods of the invention provide to the subject animprovement versus baseline in at least one of the SF-36 physical healthrelated parameters (physical health, role-physical, bodily pain and/orgeneral health) and/or in at least one of the SF-36 mental healthrelated parameters (vitality, social functioning, role-emotional and/ormental health). Such an improvement can take the form of an increase ofat least 1, for example at least 2 or at least 3 points, on the scalefor any one or more parameters.

The methods of the present invention may also improve the prognosis ofthe subject being treated. For example, the methods of the invention mayprovide to the subject a reduction in probability of a clinicalworsening event during the treatment period.

The dose of an RNAi agent that is administered to a subject may betailored to balance the risks and benefits of a particular dose, forexample, to achieve a desired level of TTR gene suppression (asassessed, e.g., based on TTR mRNA suppression, TTR protein expression,or a reduction in an amyloid deposit, as defined above) or a desiredtherapeutic or prophylactic effect, while at the same time avoidingundesirable side effects.

In one embodiment, an iRNA agent of the invention is administered to asubject as a weight-based dose. A “weight-based dose” (e.g., a dose inmg/kg) is a dose of the iRNA agent that will change depending on thesubject's weight. In another embodiment, an iRNA agent is administeredto a subject as a fixed dose. A “fixed dose” (e.g., a dose in mg) meansthat one dose of an iRNA agent is used for all subjects regardless ofany specific subject-related factors, such as weight. In one particularembodiment, a fixed dose of an iRNA agent of the invention is based on apredetermined weight or age.

Subjects can be administered a therapeutic amount of iRNA, such as about0.01 mg/kg, 0.02 mg/kg, 0.03 mg/kg, 0.04 mg/kg, 0.05 mg/kg, 0.1 mg/kg,0.15 mg/kg, 0.2 mg/kg, 0.25 mg/kg, 0.3 mg/kg, 0.35 mg/kg, 0.4 mg/kg,0.45 mg/kg, 0.5 mg/kg, 0.55 mg/kg, 0.6 mg/kg, 0.65 mg/kg, 0.7 mg/kg,0.75 mg/kg, 0.8 mg/kg, 0.85 mg/kg, 0.9 mg/kg, 0.95 mg/kg, 1.0 mg/kg, 1.1mg/kg, 1.2 mg/kg, 1.25 mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5 mg/kg, 1.6mg/kg, 1.7 mg/kg, 1.8 mg/kg, 1.9 mg/kg, 2.0 mg/kg, 2.1 mg/kg, 2.2 mg/kg,2.3 mg/kg, 2.4 mg/kg, 2.5 mg/kg dsRNA, 2.6 mg/kg dsRNA, 2.7 mg/kg dsRNA,2.8 mg/kg dsRNA, 2.9 mg/kg dsRNA, 3.0 mg/kg dsRNA, 3.1 mg/kg dsRNA, 3.2mg/kg dsRNA, 3.3 mg/kg dsRNA, 3.4 mg/kg dsRNA, 3.5 mg/kg dsRNA, 3.6mg/kg dsRNA, 3.7 mg/kg dsRNA, 3.8 mg/kg dsRNA, 3.9 mg/kg dsRNA, 4.0mg/kg dsRNA, 4.1 mg/kg dsRNA, 4.2 mg/kg dsRNA, 4.3 mg/kg dsRNA, 4.4mg/kg dsRNA, 4.5 mg/kg dsRNA, 4.6 mg/kg dsRNA, 4.7 mg/kg dsRNA, 4.8mg/kg dsRNA, 4.9 mg/kg dsRNA, 5.0 mg/kg dsRNA, 5.1 mg/kg dsRNA, 5.2mg/kg dsRNA, 5.3 mg/kg dsRNA, 5.4 mg/kg dsRNA, 5.5 mg/kg dsRNA, 5.6mg/kg dsRNA, 5.7 mg/kg dsRNA, 5.8 mg/kg dsRNA, 5.9 mg/kg dsRNA, 6.0mg/kg dsRNA, 6.1 mg/kg dsRNA, 6.2 mg/kg dsRNA, 6.3 mg/kg dsRNA, 6.4mg/kg dsRNA, 6.5 mg/kg dsRNA, 6.6 mg/kg dsRNA, 6.7 mg/kg dsRNA, 6.8mg/kg dsRNA, 6.9 mg/kg dsRNA, 7.0 mg/kg dsRNA, 7.1 mg/kg dsRNA, 7.2mg/kg dsRNA, 7.3 mg/kg dsRNA, 7.4 mg/kg dsRNA, 7.5 mg/kg dsRNA, 7.6mg/kg dsRNA, 7.7 mg/kg dsRNA, 7.8 mg/kg dsRNA, 7.9 mg/kg dsRNA, 8.0mg/kg dsRNA, 8.1 mg/kg dsRNA, 8.2 mg/kg dsRNA, 8.3 mg/kg dsRNA, 8.4mg/kg dsRNA, 8.5 mg/kg dsRNA, 8.6 mg/kg dsRNA, 8.7 mg/kg dsRNA, 8.8mg/kg dsRNA, 8.9 mg/kg dsRNA, 9.0 mg/kg dsRNA, 9.1 mg/kg dsRNA, 9.2mg/kg dsRNA, 9.3 mg/kg dsRNA, 9.4 mg/kg dsRNA, 9.5 mg/kg dsRNA, 9.6mg/kg dsRNA, 9.7 mg/kg dsRNA, 9.8 mg/kg dsRNA, 9.9 mg/kg dsRNA, 9.0mg/kg dsRNA, 10 mg/kg dsRNA, 15 mg/kg dsRNA, 20 mg/kg dsRNA, 25 mg/kgdsRNA, 30 mg/kg dsRNA, 35 mg/kg dsRNA, 40 mg/kg dsRNA, 45 mg/kg dsRNA,or about 50 mg/kg dsRNA. Values and ranges intermediate to the recitedvalues are also intended to be part of this invention.

In certain embodiments, for example, when a composition of the inventioncomprises a dsRNA as described herein and a lipid, subjects can beadministered a therapeutic amount of iRNA, such as about 0.01 mg/kg toabout 10 mg/kg, about 0.01 mg/kg to about 10 mg/kg, about 0.05 mg/kg toabout 5 mg/kg, about 0.05 mg/kg to about 10 mg/kg, about 0.1 mg/kg toabout 5 mg/kg, about 0.1 mg/kg to about 10 mg/kg, about 0.2 mg/kg toabout 5 mg/kg, about 0.2 mg/kg to about 10 mg/kg, about 0.3 mg/kg toabout 5 mg/kg, about 0.3 mg/kg to about 10 mg/kg, about 0.4 mg/kg toabout 5 mg/kg, about 0.4 mg/kg to about 10 mg/kg, about 0.5 mg/kg toabout 5 mg/kg, about 0.5 mg/kg to about 10 mg/kg, about 1 mg/kg to about5 mg/kg, about 1 mg/kg to about 10 mg/kg, about 1.5 mg/kg to about 5mg/kg, about 1.5 mg/kg to about 10 mg/kg, about 2 mg/kg to about 2.5mg/kg, about 2 mg/kg to about 10 mg/kg, about 3 mg/kg to about 5 mg/kg,about 3 mg/kg to about 10 mg/kg, about 3.5 mg/kg to about 5 mg/kg, about4 mg/kg to about 5 mg/kg, about 4.5 mg/kg to about 5 mg/kg, about 4mg/kg to about 10 mg/kg, about 4.5 mg/kg to about 10 mg/kg, about 5mg/kg to about 10 mg/kg, about 5.5 mg/kg to about 10 mg/kg, about 6mg/kg to about 10 mg/kg, about 6.5 mg/kg to about 10 mg/kg, about 7mg/kg to about 10 mg/kg, about 7.5 mg/kg to about 10 mg/kg, about 8mg/kg to about 10 mg/kg, about 8.5 mg/kg to about 10 mg/kg, about 9mg/kg to about 10 mg/kg, or about 9.5 mg/kg to about 10 mg/kg. Valuesand ranges intermediate to the recited values are also intended to bepart of this invention.

For example, the dsRNA may be administered at a dose of about 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2,3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2,6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7,7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2,9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg/kg. Values and rangesintermediate to the recited values are also intended to be part of thisinvention.

In some embodiments, for example, when a composition of the inventioncomprises a dsRNA as described herein and an N-acetylgalactosamine,subjects can be administered a therapeutic amount of iRNA, such as about0.01 mg/kg to about 5 mg/kg, about 0.01 mg/kg to about 10 mg/kg, about0.05 mg/kg to about 5 mg/kg, about 0.05 mg/kg to about 10 mg/kg, about0.15 mg/kg to about 3 mg/kg, about 0.1 mg/kg to about 5 mg/kg, about 0.1mg/kg to about 10 mg/kg, about 0.2 mg/kg to about 5 mg/kg, about 0.2mg/kg to about 10 mg/kg, about 0.3 mg/kg to about 3 mg/kg, about 0.3mg/kg to about 5 mg/kg, about 0.3 mg/kg to about 10 mg/kg, about 0.4mg/kg to about 5 mg/kg, about 0.4 mg/kg to about 10 mg/kg, about 0.5mg/kg to about 5 mg/kg, about 0.5 mg/kg to about 10 mg/kg, about 0.6mg/kg to about 3 mg/kg, about 1 mg/kg to about 3 mg/kg, about 1 mg/kg toabout 5 mg/kg, about 1 mg/kg to about 10 mg/kg, about 1.25 mg/kg toabout 3 mg/kg, about 1.5 mg/kg to about 5 mg/kg, about 1.5 mg/kg toabout 10 mg/kg, about 2 mg/kg to about 2.5 mg/kg, about 2 mg/kg to about10 mg/kg, about 3 mg/kg to about 5 mg/kg, about 3 mg/kg to about 10mg/kg, about 3.5 mg/kg to about 5 mg/kg, about 4 mg/kg to about 5 mg/kg,about 4.5 mg/kg to about 5 mg/kg, about 4 mg/kg to about 10 mg/kg, about4.5 mg/kg to about 10 mg/kg, about 5 mg/kg to about 10 mg/kg, about 5.5mg/kg to about 10 mg/kg, about 6 mg/kg to about 10 mg/kg, about 6.5mg/kg to about 10 mg/kg, about 7 mg/kg to about 10 mg/kg, about 7.5mg/kg to about 10 mg/kg, about 8 mg/kg to about 10 mg/kg, about 8.5mg/kg to about 10 mg/kg, about 9 mg/kg to about 10 mg/kg, or about 9.5mg/kg to about 10 mg/kg. Values and ranges intermediate to the recitedvalues are also intended to be part of this invention.

For example, the dsRNA may be administered at a dose of about 0.1, 0.15,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.25, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9,3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4,4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9,6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4,7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9,9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg/kg.Values and ranges intermediate to the recited values are also intendedto be part of this invention.

In other embodiments, for example, when a composition of the inventioncomprises a dsRNA as described herein and an N-acetylgalactosamine,subjects can be administered a therapeutic amount of iRNA, such as adose of about 0.1 to about 50 mg/kg, about 0.25 to about 50 mg/kg, about0.5 to about 50 mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50mg/kg, about 1.5 to about 50 mg/kg, about 2 to about 50 mg/kg, about 2.5to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50mg/kg, about 15 to about 50 mg/kg, about 20 to about 50 mg/kg, about 20to about 50 mg/kg, about 25 to about 50 mg/kg, about 25 to about 50mg/kg, about 30 to about 50 mg/kg, about 35 to about 50 mg/kg, about 40to about 50 mg/kg, about 45 to about 50 mg/kg, about 0.1 to about 45mg/kg, about 0.25 to about 45 mg/kg, about 0.5 to about 45 mg/kg, about0.75 to about 45 mg/kg, about 1 to about 45 mg/kg, about 1.5 to about 45mg/kg, about 2 to about 45 mg/kg, about 2.5 to about 45 mg/kg, about 3to about 45 mg/kg, about 3.5 to about 45 mg/kg, about 4 to about 45mg/kg, about 4.5 to about 45 mg/kg, about 5 to about 45 mg/kg, about 7.5to about 45 mg/kg, about 10 to about 45 mg/kg, about 15 to about 45mg/kg, about 20 to about 45 mg/kg, about 20 to about 45 mg/kg, about 25to about 45 mg/kg, about 25 to about 45 mg/kg, about 30 to about 45mg/kg, about 35 to about 45 mg/kg, about 40 to about 45 mg/kg, about 0.1to about 40 mg/kg, about 0.25 to about 40 mg/kg, about 0.5 to about 40mg/kg, about 0.75 to about 40 mg/kg, about 1 to about 40 mg/kg, about1.5 to about 40 mg/kg, about 2 to about 40 mg/kg, about 2.5 to about 40mg/kg, about 3 to about 40 mg/kg, about 3.5 to about 40 mg/kg, about 4to about 40 mg/kg, about 4.5 to about 40 mg/kg, about 5 to about 40mg/kg, about 7.5 to about 40 mg/kg, about 10 to about 40 mg/kg, about 15to about 40 mg/kg, about 20 to about 40 mg/kg, about 20 to about 40mg/kg, about 25 to about 40 mg/kg, about 25 to about 40 mg/kg, about 30to about 40 mg/kg, about 35 to about 40 mg/kg, about 0.1 to about 30mg/kg, about 0.25 to about 30 mg/kg, about 0.5 to about 30 mg/kg, about0.75 to about 30 mg/kg, about 1 to about 30 mg/kg, about 1.5 to about 30mg/kg, about 2 to about 30 mg/kg, about 2.5 to about 30 mg/kg, about 3to about 30 mg/kg, about 3.5 to about 30 mg/kg, about 4 to about 30mg/kg, about 4.5 to about 30 mg/kg, about 5 to about 30 mg/kg, about 7.5to about 30 mg/kg, about 10 to about 30 mg/kg, about 15 to about 30mg/kg, about 20 to about 30 mg/kg, about 20 to about 30 mg/kg, about 25to about 30 mg/kg, about 0.1 to about 20 mg/kg, about 0.25 to about 20mg/kg, about 0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg, about1 to about 20 mg/kg, about 1.5 to about 20 mg/kg, about 2 to about 20mg/kg, about 2.5 to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5to about 20 mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20mg/kg, about 5 to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10to about 20 mg/kg, or about 15 to about 20 mg/kg. In one embodiment,when a composition of the invention comprises a dsRNA as describedherein and an N-acetylgalactosamine, subjects can be administered atherapeutic amount of about 10 to about 30 mg/kg of dsRNA. Values andranges intermediate to the recited values are also intended to be partof this invention.

For example, subjects can be administered a therapeutic amount of iRNA,such as about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2,1.25, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5,2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4,4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5,5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7,7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5,8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10,10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17,17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24,24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33,34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, orabout 50 mg/kg. Values and ranges intermediate to the recited values arealso intended to be part of this invention.

In some embodiments, the RNAi agent is administered as a fixed dose ofbetween about 25 mg to about 900 mg, e.g., between about 25 mg to about850 mg, between about 25 mg to about 800 mg, between about 25 mg toabout 750 mg, between about 25 mg to about 700 mg, between about 25 mgto about 650 mg, between about 25 mg to about 600 mg, between about 25mg to about 550 mg, between about 25 mg to about 500 mg, between about100 mg to about 850 mg, between about 100 mg to about 800 mg, betweenabout 100 mg to about 750 mg, between about 100 mg to about 700 mg,between about 100 mg to about 650 mg, between about 100 mg to about 600mg, between about 100 mg to about 550 mg, between about 100 mg to about500 mg, between about 200 mg to about 850 mg, between about 200 mg toabout 800 mg, between about 200 mg to about 750 mg, between about 200 mgto about 700 mg, between about 200 mg to about 650 mg, between about 200mg to about 600 mg, between about 200 mg to about 550 mg, between about200 mg to about 500 mg, between about 300 mg to about 850 mg, betweenabout 300 mg to about 800 mg, between about 300 mg to about 750 mg,between about 300 mg to about 700 mg, between about 300 mg to about 650mg, between about 300 mg to about 600 mg, between about 300 mg to about550 mg, between about 300 mg to about 500 mg, between about 400 mg toabout 850 mg, between about 400 mg to about 800 mg, between about 400 mgto about 750 mg, between about 400 mg to about 700 mg, between about 400mg to about 650 mg, between about 400 mg to about 600 mg, between about400 mg to about 550 mg, or between about 400 mg to about 500 mg.

In some embodiments, the RNAi agent is administered as a fixed dose ofabout 12.5 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg,about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about90 mg, about 95 mg, about 100 mg, about 110 mg, about 120 mg, about 125mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170mg, about 175 mg, about 180 mg, about 190 mg, 200 mg, about 225 mg,about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg,about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg,about 500 mg, about 525 mg, about 550 mg, about 575 mg, about 600 mg,about 625 mg, about 650 mg, about 675 mg, about 700 mg, about 725 mg,about 750 mg, about 775 mg, about 800 mg, about 825 mg, about 850 mg,about 875 mg, or about 900 mg.

In certain embodiments, the RNAi agent is administered to a subject as afixed dose of about 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250,275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, orabout 600 mg once every three months (i.e., once a quarter). In oneembodiment, the administration is subcutaneous administration, e.g.,self-administration via, e.g., a pre-filled syringe or auto-injectorsyringe. In some embodiments, a dose of the RNAi agent for subcutaneousadministration is contained in a volume of less than or equal to one mlof, e.g., a pharmaceutically acceptable carrier.

In some embodiments, the RNAi agent is administered in two or moredoses. If desired to facilitate repeated or frequent infusions,implantation of a delivery device, e.g., a pump, semi-permanent stent(e.g., intravenous, intraperitoneal, intracisternal or intracapsular),or reservoir may be advisable. In some embodiments, the number or amountof subsequent doses is dependent on the achievement of a desired effect,e.g., the suppression of a TTR gene, or the achievement of a therapeuticor prophylactic effect, e.g., reducing an amyloid deposit or reducing asymptom of a TTR-associated disease.

In some embodiments, the RNAi agent is administered according to aschedule. For example, the RNAi agent may be administered twice perweek, three times per week, four times per week, or five times per week.In some embodiments, the schedule involves regularly spacedadministrations, e.g., hourly, every four hours, every six hours, everyeight hours, every twelve hours, daily, every 2 days, every 3 days,every 4 days, every 5 days, weekly, biweekly, monthly, or quarterly. Inone embodiment, a dosage of 0.3 mg/kg, 0.6 mg/kg, 1 mg/kg, 1.25 mg/kg,1.5 mg/kg, 2, 2.5 mg/kg, or 3 mg/kg is administered monthly. In anotherembodiment, a dosage of 0.3 mg/kg, 0.6 mg/kg, 1 mg/kg, 1.25 mg/kg, 1.5mg/kg, 2.2.5 mg/kg, or 3 mg/kg is administered quarterly.

In other embodiments, the schedule involves closely spacedadministrations followed by a longer period of time during which theagent is not administered. For example, the schedule may involve aninitial set of doses that are administered in a relatively short periodof time (e.g., about every 6 hours, about every 12 hours, about every 24hours, about every 48 hours, or about every 72 hours) followed by alonger time period (e.g., about 1 week, about 2 weeks, about 3 weeks,about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8weeks, about 9 weeks, about 10 weeks, about 11 weeks, or about 12 weeks)during which the RNAi agent is not administered. In one embodiment, theRNAi agent is initially administered hourly and is later administered ata longer interval (e.g., daily, weekly, biweekly, monthly, orquarterly). In another embodiment, the RNAi agent is initiallyadministered daily and is later administered at a longer interval (e.g.,weekly, biweekly, monthly, or quarterly). In certain embodiments, thelonger interval increases over time or is determined based on theachievement of a desired effect.

In a specific embodiment, the RNAi agent is administered once dailyduring a first week, followed by weekly, monthly or quarterly dosingstarting on the eighth day of administration. In another specificembodiment, the RNAi agent is administered every other day during afirst week followed by weekly, monthly or quarterly dosing starting onthe eighth day of administration. In another embodiment, the RNAi agentis administered once daily for five days during a first week, followedby weekly, monthly or quarterly dosing administration.

Any of these schedules may optionally be repeated for one or moreiterations. The number of iterations may depend on the achievement of adesired effect, e.g., the suppression of a TTR gene, retinol bindingprotein level, vitamin A level, and/or the achievement of a therapeuticor prophylactic effect, e.g., reducing an amyloid deposit or reducing asymptom of a TTR-associated disease.

In some embodiments, the RNAi agent is administered with othertherapeutic agents or other therapeutic regimens. For example, otheragents or other therapeutic regimens suitable for treating aTTR-associated disease may include a liver transplant, which can reducemutant TTR levels in the body; Tafamidis (Vyndaqel), which kineticallystabilizes the TTR tetramer preventing tetramer dissociation requiredfor TTR amyloidogenesis; and diuretics, which may be employed, forexample, to reduce edema in TTR amyloidosis with cardiac involvement.

In one embodiment, a subject is administered an initial dose and one ormore maintenance doses of an RNAi agent. The maintenance dose or dosescan be the same or lower than the initial dose, e.g., one-half of theinitial dose. A maintenance regimen can include treating the subjectwith a dose or doses ranging from 0.01 μg to 15 mg/kg of body weight perday, e.g., 1 mg/kg, 1.25 mg/kg, 1.5 mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg,4 mg/kg, 5 mg/kg, 10 mg/kg, 0.1 mg/kg, 0.15 mg/kg, 0.3 mg/kg, 0.6 mg/kg,0.01 mg/kg, 0.001 mg/kg, or 0.00001 mg/kg of bodyweight per day, or adose or doses of about 12.5 mg to about 900 mg, e.g., about 25 mg, about30 mg about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg,about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about85 mg, about 90 mg, about 95 mg, about 100 mg, about 125 mg, about 150mg, about 175 mg, 200 mg, about 225 mg, about 250 mg, about 275 mg,about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg,about 425 mg, about 450 mg, about 475 mg, about 500 mg, about 525 mg,about 550 mg, about 575 mg, about 600 mg, about 625 mg, about 650 mg,about 675 mg, about 700 mg, about 725 mg, about 750 mg, about 775 mg,about 800 mg, about 825 mg, about 850 mg, about 875 mg, or about 900 mgper week. The maintenance doses are, for example, administered no morethan once every 2 days, once every 5 days, once every 7 days, once every10 days, once every 14 days, once every 21 days, once every 30 days, oronce every 90 days. Further, the treatment regimen may last for a periodof time which will vary depending upon the nature of the particulardisease, its severity and the overall condition of the patient. Incertain embodiments the dosage may be delivered no more than once perday, e.g., no more than once per 24, 36, 48, or more hours, e.g., nomore than once every 5 or 8 days. Following treatment, the patient canbe monitored for changes in his/her condition. The dosage of the RNAiagent may either be increased in the event the patient does not respondsignificantly to current dosage levels, or the dose may be decreased ifan alleviation of the symptoms of the disease state is observed, if thedisease state has been ablated, or if undesired side-effects areobserved.

VI. Kits

The present invention also provides kits for performing any of themethods of the invention. Such kits include one or more RNAi agent(s)and instructions for use, e.g., instructions for inhibiting expressionof a TTR in a cell by contacting the cell with the RNAi agent(s) in anamount effective to inhibit expression of the TTR. The kits mayoptionally further comprise means for contacting the cell with the RNAiagent (e.g., an injection device or an infusion pump), or means formeasuring the inhibition of TTR (e.g., means for measuring theinhibition of TTR mRNA or TTR protein). Such means for measuring theinhibition of TTR may comprise a means for obtaining a sample from asubject, such as, e.g., a plasma sample. The kits of the invention mayoptionally further comprise means for administering the RNAi agent(s) toa subject or means for determining the therapeutically effective orprophylactically effective amount.

Suitable RNAi agents for inclusion in the kits of the invention includeany one of the RNAi agents listed in any one of Tables 1, 3, 5, 6, or 7.In one embodiment, the RNAi agent is selected from the group consistingof AD-66016, AD-65492, AD-66017, and AD-66018.

The RNAi agent may be provided in any convenient form, such as asolution in sterile water for injection. For example, the RNAi agent maybe provided as a 500 mg/ml, 450 mg/ml, 400 mg/ml, 350 mg/ml, 300 mg/ml,250 mg/ml, 200 mg/ml, 150 mg/ml, 100 mg/ml, or 50 mg/ml solution insterile water for injection.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references andpublished patents and patent applications cited throughout theapplication are hereby incorporated herein by reference.

EXAMPLES Example 1: In Vitro Stability and Silencing Activity ofChemically Modified RNAI Agents that Target TTR

The following experiments demonstrated the beneficial effects of certainchemical modifications, including no more than 8 2′-fluoro modificationson the sense strand, no more than 6 2′-fluoro modifications on theantisense strand, six phosphorothioate nucleotide linkages, and aligand, e.g., a GalNAc₃ ligand, on the silencing activity of RNAi agentsthat target TTR. The sequences of the agents investigated are providedin Table 1 below.

TTR siRNA sequences were synthesized at a 1 μmol scale on a Mermade 192synthesizer (BioAutomation) using the solid support mediatedphosphoramidite chemistry. The solid support was controlled pore glass(500 A) loaded with custom GalNAc ligand or universal solid support (AMbiochemical). Ancillary synthesis reagents, 2′-F and 2′-O-Methyl RNA anddeoxy phosphoramidites were obtained from Thermo-Fisher (Milwaukee,Wis.) and Hongene (China). 2′F, 2′-O-Methyl, GNA (glycol nucleic acids),5′phosphate and abasic modifications were introduced employing thecorresponding phosphoramidites. Synthesis of 3′ GalNAc conjugated singlestrands was performed on a GalNAc modified CPG support. Custom CPGuniversal solid support was used for the synthesis of antisense singlestrands. Coupling time for all phosphoramidites (100 mM in acetonitrile)was 5 minutes employing 5-Ethylthio-1H-tetrazole (ETT) as the activator(0.6 M in acetonitrile). Phosphorothioate linkages were generated usinga 50 mM solution of 3-((Dimethylamino-methylidene)amino)-3H-1,2,4-dithiazole-3-thione (DDTT, obtained from Chemgenes(Wilmington, Mass., USA)) in anhydrous acetonitrile/pyridine (1:1 v/v).Oxidation time was 3 minutes. All sequences were synthesized with finalremoval of the DMT group (“DMT off”).

Upon completion of the solid phase synthesis, oligoribonucleotides werecleaved from the solid support and deprotected in sealed 96 deep wellplates using 200 μL Aqueous Methylamine reagents at 60° C. for 20minutes. At the end of cleavage and deprotection step, the synthesisplate was allowed to come to room temperature and was precipitated byaddition of 1 mL of acetontile:ethanol mixture (9:1). The plates werecooled at −80 C for 2 hrs, supernatant decanted carefully with the aidof a multi channel pipette. The oligonucleotide pellet was re-suspendedin 20 mM NaOAc buffer and was desalted using a 5 mL HiTrap sizeexclusion column (GE Healthcare) on an AKTA Purifier System equippedwith an A905 autosampler and a Frac 950 fraction collector. Desaltedsamples were collected in 96-well plates. Samples from each sequencewere analyzed by LC-MS to confirm the identity, UV (260 nm) forquantification and a selected set of samples was analysed by IEXchromatography to determine purity.

Annealing of TTR single strands was performed on a Tecan liquid handlingrobot. Equimolar mixtures of sense and antisense single strands werecombined and annealed in 96 well plates. After combining thecomplementary single strands, the 96-well plate was sealed tightly andheated in an oven at 100° C. for 10 minutes and allowed to come slowlyto room temperature over a period 2-3 hours. The concentration of eachduplex was normalized to 10 μM in 1×PBS.

These duplexes were assayed for in vitro metabolic stability using a ratcytosol stability assay. For such an assay, female rat liver cytosol(Xenotech Cat. # R1500.C) was thawed to room temperature and diluted to1 mg/mL in 50 mM Tris buffer: HCl pH 7.4, 5 mM MgCl2. Twenty-four hoursamples were prepared by mixing 100 μL of 1 mg/mL Cytosol with 25 μL of0.4 mg/mL siRNA sample in a microcentrifuge tube and incubating for 24hours in an eppendorf Thermomixer set to 37° C. and 300 rpm. After 24hours of incubation 300 μL of Phenomenex Lysis Loading Buffer (Cat. #ALO-8498) and 12.5 μL of a 0.4 mg/mL internal standard siRNA were addedto each sample. Zero hour samples were prepared by mixing 100 μL of 1mg/mL Cytosol with 25 μL of 0.4 mg/mL siRNA sample, 300 μL of PhenomenexLysis Loading Buffer, and 12.5 μL of a 0.4 mg/mL internal standardsiRNA. siRNA was extracted from 24 hour samples and 0 hour samples usinga Phenomenex Clarity OTX Starter Kit (Cat. # KSO-8494). After thesamples were extracted they were transferred to a microcentrifuge tubeand dried down using a Labconco CentriVap Concentrator (Cat. #7810010).The samples were then resuspended with 500 μL of nuclease free water.Fifty μL of each sample was run on an Agilent Technologies 1260 InfinityBinary LC with Agilent Technologies 6130 Quadrupole LC/MS. The analysiswas run using double column setup in regeneration mode. The Quaternarypump method was run for 12.20 minutes at 0.400 mL/min with the followingtimetable:

Time Function Parameter 0.20 5% Buffer A(16 mM TEA 200 mM HFIP), 95%Buffer B (100% Methanol) 2.50 5% Buffer A(16 mM TEA 200 mM HFIP), 95%Buffer B (100% Methanol) 3.00 100% Buffer A(16 mM TEA 200 mM HFIP)The Binary Pump method was run for 12.20 min at 0.700 mL/min with thefollowing timetable:Time FunctionParameter

Time Function Parameter 0.00 100% Buffer A(16 mM TEA 200 mM HFIP) 0.40100% Buffer A(16 mM TEA 200 mM HFIP) 10.00 60% Buffer A(16 mM TEA 200 mMHFIP), 40% Buffer B (100% ACN) 10.10 100% Buffer A(16 mM TEA 200 mMHFIP) 12.20 100% Buffer A(16 mM TEA 200 mM HFIP)Both the left and right column was set at 75.00° C. The UV signal wasmeasured at 260 nm wavelength. The percent remaining of each strand wascalculated using the following equation:% Strand remaining=100*(Peak Area_(Strand 24 h)/PeakArea_(Strand 0 h)*(Peak Area_(Standard 24 h)/Peak Area_(Standard 0 h))).

The results of these twenty-four hour cytosol stability assaysdemonstrate that all of the duplexes are highly stable.

A subset of these agents was also assessed for in vitro metabolicstability using a tritosome stability assay. For the tritosome stabilityassays, rat liver tritosomes (Xenotech custom product PR14044) werethawed to room temperature and diluted to 0.5 units/mL Acid Phosphatasein 20 mM Sodium Citrate pH 5.0 Buffer. Twenty-four hour samples wereprepared by mixing 100 μL of 0.5 units/mL Acid Phosphatase Tritosomeswith 25 μL of 0.4 mg/mL siRNA sample in a microcentrifuge tube andincubating for twenty-four hours in an eppendorf Thermomixer set to 37°C. and 300 rpm. After twenty-four hours of incubation, 300 μL ofPhenomenex Lysis Loading Buffer (Cat. # ALO-8498) and 12.5 μL of a 0.4mg/mL internal standard siRNA were added to each sample. Time 0 hoursamples were prepared by mixing 100 μL of 0.5 units/mL Acid PhosphataseTritosomes with 25 μL of 0.4 mg/mL siRNA sample, 300 μL of PhenomenexLysis Loading Buffer, and 12.5 μL of a 0.4 mg/mL internal standardsiRNA. siRNA was extracted from twenty-four hour samples and 0 hoursamples using a Phenomenex Clarity OTX Starter Kit (Cat. # KSO-8494).After the samples were extracted, they were transferred to amicrocentrifuge tube and dried down using a Labconco CentriVapConcentrator (Cat. #7810010). The samples were then resuspended with 500μL of nuclease free water. Fifty μL of each sample was run on an AgilentTechnologies 1260 Infinity Binary LC with Agilent Technologies 6130Quadrupole LC/MS. The analysis was run using double column setup inregeneration mode. The Quaternary pump method was run for 12.20 minutesat 0.400 mL/min with the following timetable:

Time Function Parameter 0.20 5% Buffer A(16 mM TEA 200 mM HFIP), 95%Buffer B (100% Methanol) 2.50 5% Buffer A(16 mM TEA 200 mM HFIP), 95%Buffer B (100% Methanol) 3.00 100% Buffer A(16 mM TEA 200 mM HFIP)The Binary Pump method was run for 12.20 min at 0.700 mL/min with thefollowing timetable:

Time Function Parameter 0.00 100% Buffer A(16 mM TEA 200 mM HFIP) 0.40100% Buffer A(16 mM TEA 200 mM HFIP) 10.00 60% Buffer A(16 mM TEA 200 mMHFIP), 40% Buffer B (100% ACN) 10.10 100% Buffer A(16 mM TEA 200 mMHFIP) 12.20 100% Buffer A(16 mM TEA 200 mM HFIP)Both the left and right column was set at 75.00° C. The UV signal wasmeasured at 260 nm wavelength. The percent remaining of each strand wascalculated using the following equation:% Strand remaining=100*(Peak Area_(Strand 24 h)/PeakArea_(Strand 0 h)*(Peak Area_(Standard 24 h)/Peak Area_(Standard 0 h))).

The results of the twenty-four hour tritosome stability assays, providedin FIG. 1, demonstrate that all of the duplexes are highly stable intritosomes.

TABLE 1 Modified Sense and Antisense Strand Sequences of TTR dsRNAs SEQSense sequence ID Duplex ID Sense ID 5′ to 3′ NO Antisense ID AD-51547A-106305 UfgGfgAfuUfuCfAfUfgUfaacCfaAfgAfL96 15 A-102978 AD-58142A-117240 UfsgsGfgAfuUfuCfAfUfgUfaacCfaAfgAfL96 16 A-117241 AD-64527A-128512 usgsggauuucadTguaacaaagaL96 17 A-128525 AD-65367 A-128499usgsggAfuUfuCfAfUfgUfaaccaagAfL96 18 A-128520 AD-65489 A-131365usgggauuucadTguaacaaagaL96 19 A-128520 AD-65481 A-131354usgsggauUfuCfAfUfguaaccaagaL96 20 A-131364 AD-65488 A-131354usgsggauUfuCfAfUfguaaccaagaL96 21 A-131358 AD-65496 A-131354usgsggauUfuCfAfUfguaaccaagaL96 22 A-131360 AD-65491 A-128557usgsggauuucadTguaacY34aagaL96 23 A-128525 AD-65495 A-131353usgsggauUfuCfAfUfguaaCfcaagaL96 24 A-128516 AD-65367 A-128499usgsggAfuUfuCfAfUfgUfaaccaagAfL96 25 A-128520 AD-65493 A-128512usgsggauuucadTguaacaaagaL96 26 A-131366 AD-65494 A-128512usgsggauuucadTguaacaaagaL96 27 A-128526 AD-64527 A-128512usgsggauuucadTguaacaaagaL96 28 A-128525 SEQ Antisense sequence ID DuplexID 5′ to 3′ NO AD-51547 uCfuUfgGfUfUfaCfaugAfaAfuCfcCfasUfsc 29 AD-58142usCfsuUfgGfUfUfaCfaugAfaAfuCfcCfasusc 30 AD-64527usdCsuugguuadCaugdAaaucccasusc 31 AD-65367 usCfsuugguuacaugAfaaucccasusc32 AD-65489 usCfsuugguuacaugAfaaucccasusc 33 AD-65481UfsCfsuugGfuuacaugAfaAfucccasusc 34 AD-65488PusCfsuugGfuuacaugAfaAfucccasusc 35 AD-65496PusCfsuugGfuuAfcaugAfaAfucccasusc 36 AD-65491usdCsuugguuadCaugdAaaucccasusc 37 AD-65495usCfsuugGfuUfAfcaugAfaAfucccasusc 38 AD-65367usCfsuugguuacaugAfaaucccasusc 39 AD-65493 PusCfsuugguuacaugAfaaucccasusc40 AD-65494 PusdCsuugguuadCaugdAaaucccasusc 41 AD-64527usdCsuugguuadCaugdAaaucccasusc 42

TABLE 2 Abbreviations of nucleotide monomers used in nucleic acidsequence representation. It will be understood that these monomers, whenpresent in an oligonucleotide, are mutually linked by5′-3′-phosphodiester bonds. Abbreviation Nucleotide(s) AAdenosine-3′-phosphate Af 2′-fluoroadenosine-3′-phosphate Afs2′-fluoroadenosine-3′-phosphorothioate As adenosine-3′-phosphorothioateC cytidine-3′-phosphate Cf 2′-fluorocytidine-3′-phosphate Cfs2′-fluorocytidine-3′-phosphorothioate Cs cytidine-3′-phosphorothioate Gguanosine-3′-phosphate Gf 2′-fluoroguanosine-3′-phosphate Gfs2′-fluoroguanosine-3′-phosphorothioate Gs guanosine-3′-phosphorothioateT 5′-methyluridine-3′-phosphate Tf2′-fluoro-5-methyluridine-3′-phosphate Tfs2′-fluoro-5-methyluridine-3′-phosphorothioate Ts5-methyluridine-3′-phosphorothioate U Uridine-3′-phosphate Uf2′-fluorouridine-3′-phosphate Ufs 2′-fluorouridine-3′-phosphorothioateUs uridine-3′-phosphorothioate N any nucleotide (G, A, C, T or U) a2′-O-methyladenosine-3′-phosphate as2′-O-methyladenosine-3′-phosphorothioate c2′-O-methylcytidine-3′-phosphate cs2′-O-methylcytidine-3′-phosphorothioate g2′-O-methylguanosine-3′-phosphate gs2′-O-methylguanosine-3′-phosphorothioate t2′-O-methyl-5-methylthymine-3′-phosphate ts2′-O-methyl-5-methylthymine-3′-phosphorothioate u2′-O-methyluridine-3′-phosphate us2′-O-methyluridine-3′-phosphorothioate s phosphorothioate linkage L96N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4- hydroxyprolinolHyp-(GalNAc-alkyl)3 dA deoxy-adenosine dC deoxy-cytodine dGdeoxy-guanosine (dT) 2′-deoxythymidine-3′-phosphate Y342-hydroxymethyl-tetrahydrofurane-4-methoxy-3- phosphate (abasic 2′-OMefuranose) Y44 2-hydroxymethyl-tetrahydrofurane-5-phosphate (Cgn)Cytidine-glycol nucleic acid (GNA) P Phosphate VP Vinyl-phosphate

An additional set of agents targeting TTR were designed and synthesized.The sequences of these agents are provided in Table 3, below.

These additional agents were evaluated in in vitro assays. Inparticular, the IC₅₀ for each iRNA agent was determined in Hep3B cells(a human hepatoma cell line) or primary cynomologous hepatocytes (LifeTechnologies) by standard reverse transfection using LipofectamineRNAiMAX. Hep3b cells were cultured in EMEM with 10% FBS, while primarycynomologous hepatocytes were thawed immediately prior to use andcultured in WMEM with 10% FBS. Reverse transfection was carried out byadding 5 μL of RNA duplex per well into a 384-well plate along with 4.9μL of Opti-MEM plus 0.1 μL of Lipofectamine RNAiMax per well(Invitrogen, Carlsbad Calif. cat #13778-150) and incubating at roomtemperature for 15-20 minutes. Following incubation, 40 μL of completegrowth media without antibiotic containing 5,000 Hep3B cells or primarycynomologous hepatocytes was then added to each well. Collagen-coatedplates were used for the primary hepatocytes. Cells were incubated for24 hours at 37° C. in an atmosphere of 5% CO₂ prior to lysis andanalysis of TTR and GAPDH mRNA by RT-qPCR. Eight different siRNAconcentrations ranging from 10 nM to 0.38 fM were assessed for IC₅₀determination and TTR/GAPDH for siRNA transfected cells was normalizedto cells transfected with 10 nM Luciferase siRNA.

Free uptake silencing in primary cynomolgus hepatocytes was assessedfollowing incubation with TTR siRNA for 24 hours. The method was similarto that described above, with the exception that 5 μL complete growthmedium was substituted for the 5 μL containing Lipofectamine RNAiMax andOptimem. Downstream analysis for TTR and GAPDH mRNA was performed asdescribed above. For a typical dose response curve, siRNAs were titratedfrom 500 nM to 0.1.8 pM by eight-point 6-fold serial dilution.

The results of these assays (provided in Table 4) demonstrate that allof the duplexes potently inhibit TTR mRNA expression.

The in vitro stability of these additional agents was also assessedusing the cytosol and tritosome stability assays described above.

The results of the twenty-four hour cytosol stability assay are providedin FIG. 2A and the results of the twenty-four hour tritosome stabilityassay are provided in FIG. 2B and demonstrate that all of the duplexesare highly stable in tritosomes and rat cytosol.

TABLE 3 Modified Sense and Antisense Strand Sequences of TTR dsRNAs SEQDuplex Sense sequence SEQ ID Antisense Antisense sequence ID ID Sense ID5′ to 3′ NO ID 5′ to 3′ NO AD-66016 A-131354usgsggauUfuCfAfUfguaaccaagaL96 43 or A-128520usCfsuugguuacaugAfaaucccasusc 47 10 or 6 AD-65492 A-131354usgsggauUfuCfAfUfguaaccaagaL96 44 or A-131359usCfsuugGfuuAfcaugAfaAfucccasusc 48 10 or 7 AD-66017 A-131354usgsggauUfuCfAfUfguaaccaagaL96 45 or A-131903UfsCfsuugGfuuAfcaugAfaAfucccasusc 49 10 or 8 AD-66018 A-131354usgsggauUfuCfAfUfguaaccaagaL96 46 or A-131902VPusCfsuugGfuuAfcaugAfaAfucccasusc 50 10 or 9

TABLE 4 In vitro Activity of Additional TTR RNAi Agents AD-66016 (65367AS) AD-65492 (u) AD-66017 (Uf) AD-66018 (VPu) IC50 (nM) TransfectionFree Uptake Transfection Free Uptake Transfection Free UptakeTransfection Free Uptake Hep3b 0.931 N/A 0.722 N/A 0.108 N/A 0.053 N/ACyno Hepatocytes 0.235 5.157 0.21 3.629 0.026 0.284 0.015 0.191

Example 2. In Vivo TTR Silencing

The in vivo efficacy of the additional agents described above wasassessed in transgenic mice that express the valine 30 methioninevariant of human TTR (V30M hTTR) (see, e.g., Nagata, et al. (1995) JBiochem. 117:169-175; Kohno, et al. (1997) Am J Pathol.150(4):1497-508). The V30M variant of TTR is known to cause familialamyloid polyneuropathy type I in humans. See, e.g., Lobato, L. (2003) JNephrol., 16(3):438-42.

Eleven- to thirteen-month old TTR V30m mice were subcutaneouslyadministered a single 1 mg/kg or 2.5 mg/kg dose of the agents and thelevel of TTR was determined in the serum of the animals pre-dose and atdays 3, 7, 10, 14, 21, 28, 35, 42, 56, 70 and 84 post-dose. Briefly, TTRlevels were assayed using a validated TTR enzyme-linked immunosorbentassay (ELISA) (see, e.g., Coelho, et al. (2013) N Engl J Med 369:819).Ninety-six-well immuno-microplates were coated at 4° with rabbitanti-human TTR pAb (Abcam) 24 hours prior to the start of the TTR SerumProtein ELISA assay. On the day of assay, plates were washed in TBS-T,and blocked in 1× Powerblock (Biogenex) for 2 hours at room temperature.Serum samples were diluted 15,000 fold in 1× Powerblock. A 12-pointhuman TTR standard curve employing a human TTR protein standard(Sigma-Aldrich, P1742), was generated using 1.6× serial dilutions,ranging from 250 to 0 ng/mL. Following the block, 100 μL volumes ofstandards and samples were added to the plate and allowed to incubatefor 2 hours at room temperature. Plates were washed in TBS-T, andincubated for 1 hour at room temperature with Sheep Anti-hTTR primaryantibody (AbCam) diluted 1:2500 in 1× Powerblock. After a TBS-T wash,plates were incubated for 1 hour at room temperature with Donkeyanti-sheep-Alkaline phosphatase secondary antibody (AbCam) diluted3:10000 in 1× Powerblock. Plates were washed in TBS-T and 100 μL volumeof prepared substrate (SIGMAFAST™ p-Nitrophenyl phosphate Tablets) wasadded per well and allowed to react for 30 minutes at room temperaturein the dark. Reactions were quenched with 0.05 ml per well of 1 M NaOH.Absorbance at 405 nm was read on a SpectraMax plate reader, and datawere fit to a 4-parameter curve to determine serum TTR protein levels inμg/mL. Protein levels from individual animals were normalized to theirrespective individual pre-dose plasma protein values to determine thefraction TTR remaining relative to pre-dose.

The results of the 1 mg/kg single dose experiments are provided in FIG.3 and the results of the 2.5 mg/kg single dose experiments are providedin FIG. 4. The results demonstrate that all of the agents potently anddurably inhibit TTR expression, with a nadir reached at about day 7post-administration. The results also demonstrate that at day 42following a single 1 mg/kg dose of AD-65492, AD-66017, or AD-66018, morethan 40% serum TTR suppression remains, and at day 42 following a single2.5 mg/kg dose of AD-65492 or AD-66018, more than 60% serum TTRsuppression remains. Recovery to baseline serum TTR concentrationsoccurs between 56 and 84 days post-administration following a single 1mg/kg dose, and between days 70 and 84 post-administration following asingle 2.5 mg/kg dose.

The effective dose for 80% silencing in the animals (ED₈₀) wascalculated as 1 mg/kg. These data, thus, indicate that AD-65492,AD-66016, AD-66017, and AD-66018 are effective for treating a subjecthaving a TTR-associated disorder in low dose and/or monthly dosingregimes.

Example 3. Rat Exploratory Toxicity Study

Preclinical toxicity studies of AD-66016, AD-65492, AD-66017, andAD-66018 were also performed in rats. Briefly, at days 1, 8, and 15,five male rats per group were subcutaneously administered either a 30mg/kg or 100 mg/kg dose of AD-66016, AD-65492, AD-66017, or AD-66018.Control animals were administered a placebo at days 1, 8, and 15. At day16, all of the animals were sacrificed. Prior to sacrifice, the animalswere monitored daily for any clinical symptoms and body weights weremeasured weekly. After sacrifice, the animals were necropsied, andsamples were analyzed by complete serum chemistry, hematology andcoagulation panels, by histopathology of the liver and kidney, and forliver transaminase concentration.

The results of these analyses demonstrate that AD-66016, AD-65492,AD-66017, and AD-66018 are well-tolerated clinically with no adverseclinical signs or body weight changes.

Example 4. Efficacy of Multi-Dose Administration of AD-65492 andAD-66017

The effect of a multi-dose regimen of AD-65492 and AD-66017 on TTRprotein expression in hTTR V30M transgenic (Tg) mice was evaluated.

In one set of experiments, eleven- to thirteen-month old hTTR V30M micewere subcutaneously administered a weekly 2 mg/kg dose of AD-65492 forthree weeks (QW×3) and the level of TTR protein was determined in theserum of the animals pre-dose and at days 7, 14, 17, and 21 post-dose,as described above.

FIG. 5 demonstrates that administration of AD-65492 in a QW×3 dosingregimen to hTTR V30M Tg mice achieved and sustained a greater than 90%suppression of serum TTR protein expression.

In another set of experiments, AD-65492 and AD-66017 were subcutaneouslyadministered to eleven- to thirteen-month old hTTR V30M Tg mice at a 0.3mg/kg dose once a month for four months (QM×4 @ 0.3 mg/kg), at a 1 mg/kgdose once a month for four months (QM4 @ 1 mg/kg), or at a 3 mg/kg doseonce a month for four months (QM4 @ 3 mg/kg). Serun TTR protein levelswere determined as described above pre-dose and at days 7, 14, 21, 28,35, 42, 49, 56, 63, 70, 84, 91, 98, and 185 post-dose.

As shown in FIGS. 6A-6C, the TTR knockdown levels at day 7 post-dosewere similar post-second, third and fourth doses and the nadir of TTRexpression achieved at the 3 mg/kg, 1 mg/kg and 0.3 mg/kg dose isgreater than 80%, about 70-85%, and about 25-35%, respectively. Thesegraphs also demonstrate that AD-65492 provides a more sustained level ofTTR silencing than AD-66017, to more than 100 days post final dose, withabout 60%, 40%, and about 35% remaining TTR suppression at 3 mg/kg, 1mg/kg, and 0.3 mg/kg, respectively. Furthermore, for AD-65492,multi-dosing (QM×4) is additive at the 0.3 mg/kg dose resulting in about40% TTR knockdown after the fourth monthly dose as compared to the firstdose having a knockdown of about 25-35%. In addition, although there wassome recovery of TTR protein levels prior to each monthly dose acrossall dose levels for each agent, like a single subcutaneous dose, theeffective dose to achieve 80% knockdown (ED₈₀) for the multi-doseregimens was calculated as about 1 mg/kg. Thus, the pharmacodynamicactivity and kinetics of both compounds in all three dosing regimens wascomparable to the pharmacodynamic activity and kinetics of the samecompounds when administered as a single dose.

Example 5. TTR Silencing in Non-Human Primates

As demonstrated in the Examples above, AD-65492 and AD-66017 arewell-tolerated and potently and durably suppress TTR protein levels invivo.

Accordingly, the efficacy of AD-65492 and AD-66017 was further studiedby administration of different doses and different dosing regimens ofthese iRNA agents in Cynomologous monkeys. FIG. 7 provides an outline ofthis study. Briefly, four Groups, Groups 1, 2, 4, and 5, weresubcutaneously administered either a single 0.3 mg/kg dose (Groups 1 and4) or a single 1 mg/kg dose of the iRNA agent (Groups 2 and 5). Fourother Groups, Groups 3 and 6-8, were administered a monthly dose ofeither 1 mg/kg for 4 months (QM×4) (Groups 7 and 8) or a monthly dose of3 mg/kg for 4 months (QM×4) (Groups 3 and 6). Serum was collected onDays −7 and −1 pre-dose and days 3, 7, 10, 14, 21, 28, 35, 42, 49, 56,63, 70, 77, 84, 91, 105, and 119 post-dose; plasma was collected on day1 pre-dose, and 0.5, 1, 2, 4, 8, 24, 48, 96 and 168 hours post dose. Theserum level of TTR protein was determined as described above.

FIG. 8A provides the results of the 0.3 mg/kg single dose study, FIG. 8Bprovides the results of the 1 mg/kg single dose study, and FIG. 8Cprovides the results of the 3 mg/kg single dose study. For comparisonpurposes, FIG. 8B also depicts the effect of administration of a singlesubcutaneous 2.5 mg/kg dose of AD-51547 on TTR expression inCynomologous monkeys, and FIG. 8C also depicts the effect ofadministration of a single subcutaneous 5 mg/kg dose of AD-51547 on TTRexpression in Cynomologous monkeys. These graphs demonstrate that theED₅₀ for both AD-65492 and AD-66017 iRNA agents is about 0.3 mg/kg, thatthe nadir of TTR expression is reached at about day 28 for both dosesand for both iRNA agents, and that AD-65492 is more effective atsuppressing TTR protein expression than AD-66017 at the higher doselevel with a single dose. The graphs also demonstrate that AD-65942provides greater than 90% sustained suppression of TTR expressionthrough Day 42 following a single 1 mg/kg dose and greater than 80% TTRsuppression through Day 63, with about 40% remaining TTR suppression byday 119 post-dose, and that AD-66017 provides a maximum of 73%suppression of TTR expression following a single 1 mg/kg dose, withrecovery of TTR expression beginning before Day 35 and recovery towithin about 20% of baseline by day 119 post-dose.

FIG. 9A provides the results of the 1 mg/kg multi-dose study and FIG. 9Bprovides the results of the 3 mg/kg multi-dose study for AD-65492 andAD-66017. For comparison purposes, FIG. 9A also depicts the effect ofthe administration of a daily 5 mg/kg dose of AD-51547 for 5 days (firstfive arrows at days 0-4), followed by a weekly 5 mg/kg dose for fourweeks (arrows at days 7, 14, 21, and 28) (QD×5, QW×4) on TTR expressionin Cynomologous monkeys. The graphs demonstrate that both iRNA agentsprovide robust TTR protein suppression and that both iRNA agentscompletely suppress TTR protein expression between Days 21 and 28 at the3 mg/kg dose. The graphs also demonstrate that AD-65492 is moreefficacious that AD-66017 at the 1 mg/kg dose. In addition, the graphsdemonstrate that the nadir of TTR expression is achieved between Days 35and 42 for both iRNA agents at 1 mg/kg, with greater than 85%suppression prior to the second monthly dose of AD-65492, and about 70%suppression prior to the second monthly dose of AD-66017. Maintenance ofabout 95% and 85% suppression following the third and fourth monthlydoses for AD-65492 and AD-66017, respectively, was achieved.

Furthermore, as demonstrated in FIG. 10A, monthly dosing (QM×4) ofAD-65492 results in maintenance of greater than 95% TTR suppression and,as demonstrated in FIG. 10B, multi-dosing (QM×4) of AD-66017 is additiveat the 1 mg/kg dose. FIG. 10B also demonstrates that there is 85%suppression of TTR protein expression following the second monthly doseof AD-66017 and that this suppression is maintained with the third andfourth monthly doses.

Example 6. Design and Synthesis of Chemically Modified Agents TargetingTTR

Additional double stranded RNAi agents targeting TTR in whichsubstantially all of the sense strand nucleotides and substantially allof the antisense strand nucleotides are modified nucleotides andcomprising an antisense strand comprising a region of complementary toSEQ ID NO:2 were designed and synthesized as described above.

The nucleotide sequences of the sense and antisense strands of theseagents are provided in Table 5.

Example 7. Design and Synthesis of Chemically Modified Agents TargetingTTR

Additional double stranded RNAi agents targeting TTR were designed andsynthesized as described above.

The nucleotide sequences of the unmodified sense and antisense strandsof these agents are provided in Table 6, and the nucleotide sequences ofthe modified sense and antisense strands of these agents are provided inTable 7.

TABLE 5 Modified Sense and Antisense Strand Sequences of TTR dsRNAsSense SEQ SEQ strand Sense sequence ID Antisense Antisense sequence IDDuplexID ID 5′-3′ NO ID 5′-3′ NO AD-65496 A-131354usgsggauUfuCfAfUfguaaccaagaL96 51 A-131360PusCfsuugGfuuAfcaugAfaAfucccasusc 93 AD-65488 A-131354usgsggauUfuCfAfUfguaaccaagaL96 52 A-131358PusCfsuugGfuuacaugAfaAfucccasusc 94 AD-65474 A-131354usgsggauUfuCfAfUfguaaccaagaL96 53 A-131362PusCfsuugGfuuAfcaugAfAfaucccasusc 95 AD-65478 A-131354usgsggauUfuCfAfUfguaaccaagaL96 54 A-131363UfsCfsuugGfuuAfcaugAfAfaucccasusc 96 AD-65493 A-128512usgsggauuucadTguaacaaagaL96 55 A-131366 PusCfsuugguuacaugAfaaucccasusc97 AD-65481 A-131354 usgsggauUfuCfAfUfguaaccaagaL96 56 A-131364UfsCfsuugGfuuacaugAfaAfucccasusc 98 AD-65489 A-131365usgggauuucadTguaacaaagaL96 57 A-128520 usCfsuugguuacaugAfaaucccasusc 99AD-65495 A-131353 usgsggauUfuCfAfUfguaaCfcaagaL96 58 A-128516usCfsuugGfuUfAfcaugAfaAfucccasusc 100 AD-65482 A-131373usasggauUfuCfAfUfguaaccaagaL96 59 A-131374usCfsuugGfuuacaugAfaAfuccuasusu 101 AD-65468 A-131354usgsggauUfuCfAfUfguaaccaagaL96 60 A-128516usCfsuugGfuUfAfcaugAfaAfucccasusc 102 AD-65367 A-128499usgsggAfuUfuCfAfUfgUfaaccaagAfL96 61 A-128520usCfsuugguuacaugAfaaucccasusc 103 AD-65485 A-128512usgsggauuucadTguaacaaagaL96 62 A-128520 usCfsuugguuacaugAfaaucccasusc104 AD-65470 A-131367 gsgsauUfuCfAfUfguaaccaagaL96 63 A-131369usCfsuugguuacaugAfaauccscsa 105 AD-65469 A-131354usgsggauUfuCfAfUfguaaccaagaL96 64 A-131361usCfsuugGfuuAfcaugAfAfaucccasusc 106 AD-65473 A-131355usgggauUfuCfAfUfguaaccaagaL96 65 A-131356usCfuugGfuUfAfcaugAfaAfucccasusc 107 AD-65484 A-131355usgggauUfuCfAfUfguaaccaagaL96 66 A-131357 usCfuugGfuuacaugAfaAfucccasusc108 AD-65477 A-131353 usgsggauUfuCfAfUfguaaCfcaagaL96 67 A-128517usCfsuugGfuuacaugAfaAfucccasusc 109 AD-65497 A-131367gsgsauUfuCfAfUfguaaccaagaL96 68 A-131368 usCfsuugguuacaugAfaauccsusa 110AD-65475 A-131370 gsgauUfuCfAfUfguaaccaagaL96 69 A-131371usCfuugguuacaugAfaauccscsa 111 AD-65480 A-131354usgsggauUfuCfAfUfguaaccaagaL96 70 A-128517usCfsuugGfuuacaugAfaAfucccasusc 112 AD-65479 A-131372gsgsauuucAfUfguaaccaagaL96 71 A-131369 usCfsuugguuacaugAfaauccscsa 113AD-65492 usgsggauUfuCfAfUfguaaccaagaL96 72usCfsuugGfuuAfcaugAfaAfucccasusc 114 AD-65494 A-1285120usgsggauuucadTguaacaaagaL96 73 A-128526 PusdCsuugguuadCaugdAaaucccasusc115 AD-65499 A-128557 usgsggauuucadTguaacY34aagaL96 74 A-128526PusdCsuugguuadCaugdAaaucccasusc 116 AD-65491 A-128557usgsggauuucadTguaacY34aagaL96 75 A-128525 usdCsuugguuadCaugdAaaucccasusc117 AD-65498 A-1285121 usgsggauuucadTguaacaaagaL96 76 A-131375UfsdCsuugguuadCaugdAaaucccasusc 118 AD-65490 A-128512usgsggauuucadTguaacaaagaL96 77 A-128553 usdCsuugguuadCsaugdAsaaucccasusc119 AD-64520 A-128557 usgsggauuucadTguaacY34aagaL96 78 A-128553usdCsuugguuadCsaugdAsaaucccasusc 120 AD-64527 A-128512usgsggauuucadTguaacaaagaL96 79 A-128525 usdCsuugguuadCaugdAaaucccasusc121 AD-65472 A-131376 gsgsauuucadTguaacaaagaL96 80 A-131377usdCsuugguuadCaugdAaauccscsa 122 AD-65486 A-128557usgsggauuucadTguaacY34aagaL96 81 A-128520 usCfsuugguuacaugAfaaucccasusc123 AD-65472 A-131376 gsgsauuucadTguaacaaagaL96 82 A-131377usdCsuugguuadCaugdAaauccscsa 124 AD-64515usgsggauuucadTguaac(Cgn)aagaL96 83 usdCsuugguuadCaugdAaaucccasusc 125AD-64536 usgsggauuucadTguaac(Cgn)aagaL96 84usdCsuugguuadCsaugdAsaaucccasusc 126 AD-65471usgsggauuucadTguaac(Cgn)aagaL96 85 usCfsuugguuacaugAfaaucccasusc 127AD-65483 usgsggauuucadTguaac(Cgn)aagaL96 86PusdCsuugguuadCaugdAaaucccasusc 128 AD-59152 A-106305UfgGfgAfuUfuCfAfUfgUfaacCfaAfgAf 87 A-119923PuCfuUfgGfUfUfaCfaugAfaAfuCfcCfas 129 L96 Ufsc AD-65476 A-106305UfgGfgAfuUfuCfAfUfgUfaacCfaAfgAf 88 A-131351UfCfuUfgGfUfUfaCfaugAfaAfuCfcCfas 130 L96 Ufsc AD-64480 A-128493UfsgsGfgAfuUfuCfAfUfgUfaAfcCfaAf 89 A-128495PusCfsuUfgGfuUfaCfaugAfaAfuCfcCfas 131 gAfL96 usc AD-51547 A-106305UfgGfgAfuUfuCfAfUfgUfaacCfaAfgAf 90 A-1029782uCfuUfgGfUfUfaCfaugAfaAfuCfcCfasU 132 L96 fsc AD-65487 A-1284937UfsgsGfgAfuUfuCfAfUfgUfaAfcCfaAf 91 A-131352UfsCfsuUfgGfuUfaCfaugAfaAfuCfcCfas 133 gAfL96 usc AD-64474 A-1284935UfsgsGfgAfuUfuCfAfUfgUfaAfcCfaAf 92 A-1284947usCfsuUfgGfuUfaCfaugAfaAfuCfcCfasu 134 gAfL96 sc

TABLE 6 Unmodified Sense and Antisense Strand Sequences of TTR dsRNAsAntisense Start SEQ Site Relative SEQ ID to ID Duplex ID Sense sequence(5′ to 3′) NO NM_000371.2 Antisense sequence (5′ to 3′) NO AD-68322AUGGGAUUUCAUGUAACCAAA 135 504 UUUGGUUACAUGAAAUCCCAUCC 167 AD-60668AUGGGAUUUCAUGUAACCAAA 136 504 UUUGGUUACAUGAAAUCCCAUCC 168 AD-68330AUGGGAUUUCAUGUAACCAAA 137 504 UUUGGUUACAUGAAAUCCCAUCC 169 AD-64474UGGGAUUUCAUGUAACCAAGA 138 505 UCUUGGUUACAUGAAAUCCCAUC 170 AD-65468UGGGAUUUCAUGUAACCAAGA 139 505 UCUUGGUUACAUGAAAUCCCAUC 171 AD-65492UGGGAUUUCAUGUAACCAAGA 140 505 UCUUGGUUACAUGAAAUCCCAUC 172 AD-65480UGGGAUUUCAUGUAACCAAGA 141 505 UCUUGGUUACAUGAAAUCCCAUC 173 AD-60636UUUCAUGUAACCAAGAGUAUU 142 510 AAUACUCUUGGUUACAUGAAAUC 174 AD-68320UUUCAUGUAACCAAGAGUAUU 143 510 AAUACUCUUGGUUACAUGAAAUC 175 AD-68326UUUCAUGUAACCAAGAGUAUU 144 510 AAUACUCUUGGUUACAUGAAAUC 176 AD-60611UGUAACCAAGAGUAUUCCAUU 145 515 AAUGGAAUACUCUUGGUUACAUG 177 AD-68331UGUAACCAAGAGUAUUCCAUU 146 515 AAUGGAAUACUCUUGGUUACAUG 178 AD-68315UGUAACCAAGAGUAUUCCAUU 147 515 AAUGGAAUACUCUUGGUUACAUG 179 AD-68319AACCAAGAGUAUUCCAUUUUU 148 518 AAAAAUGGAAUACUCUUGGUUAC 180 AD-60612AACCAAGAGUAUUCCAUUUUU 149 518 AAAAAUGGAAUACUCUUGGUUAC 181 AD-68316AACCAAGAGUAUUCCAUUUUU 150 518 AAAAAUGGAAUACUCUUGGUUAC 182 AD-60664UUUUUACUAAAGCAGUGUUUU 151 534 AAAACACUGCUUUAGUAAAAAUG 183 AD-68321UUUUUACUAAAGCAGUGUUUU 152 534 AAAACACUGCUUUAGUAAAAAUG 184 AD-68318UUUUUACUAAAGCAGUGUUUU 153 534 AAAACACUGCUUUAGUAAAAAUG 185 AD-60665UUACUAAAGCAGUGUUUUCAA 154 537 UUGAAAACACUGCUUUAGUAAAA 186 AD-60642CUAAAGCAGUGUUUUCACCUA 155 540 UAGGUGAAAACACUGCUUUAGUA 187 AD-68329CUAAAGCAGUGUUUUCACCUA 156 540 UAGGUGAAAACACUGCUUUAGUA 188 AD-68334CUAAAGCAGUGUUUUCACCUA 157 540 UAGGUGAAAACACUGCUUUAGUA 189 AD-68328GGCAGAGACAAUAAAACAUUA 158 582 UAAUGUUUUAUUGUCUCUGCCUG 190 AD-68333GGCAGAGACAAUAAAACAUUA 159 582 UAAUGUUUUAUUGUCUCUGCCUG 191 AD-60639GGCAGAGACAAUAAAACAUUA 160 582 UAAUGUUUUAUUGUCUCUGCCUG 192 AD-60643CAGAGACAAUAAAACAUUCCU 161 584 AGGAAUGUUUUAUUGUCUCUGCC 193 AD-68317CAGAGACAAUAAAACAUUCCU 162 584 AGGAAUGUUUUAUUGUCUCUGCC 194 AD-68335CAGAGACAAUAAAACAUUCCU 163 584 AGGAAUGUUUUAUUGUCUCUGCC 195 AD-68327CAAUAAAACAUUCCUGUGAAA 164 590 UUUCACAGGAAUGUUUUAUUGUC 196 AD-68332CAAUAAAACAUUCCUGUGAAA 165 590 UUUCACAGGAAUGUUUUAUUGUC 197 AD-60637CAAUAAAACAUUCCUGUGAAA 166 590 UUUCACAGGAAUGUUUUAUUGUC 198

TABLE 7 Modified Sense and Antisense Strand Sequences of TTR dsRNAs SEQSEQ ID ID Duplex ID Sense sequence (5′ to 3′) NO Antisense sequence(5′ to 3′) NO AD-68322 asusgggaUfuUfCfAfuguaaccaaaL96 199usUfsuggUfuAfCfaugaAfaUfcccauscsc 231 AD-60668AfsusGfgGfaUfuUfCfAfuGfuAfaCfcAfaAfL96 200usUfsuGfgUfuAfcAfugaAfaUfcCfcAfuscsc 232 AD-68330asusgggaUfuUfCfAfuguaaccaaaL96 201 usUfsuggUfuacaugaAfaUfcccauscsc 233AD-64474 UfsgsGfgAfuUfuCfAfUfgUfaAfcCfaAfgAfL96 202usCfsuUfgGfuUfaCfaugAfaAfuCfcCfasusc 234 AD-65468usgsggauUfuCfAfUfguaaccaagaL96 203 usCfsuugGfuUfAfcaugAfaAfucccasusc 235AD-65492 usgsggauUfuCfAfUfguaaccaagaL96 204usCfsuugGfuuAfcaugAfaAfucccasusc 236 AD-65480usgsggauUfuCfAfUfguaaccaagaL96 205 usCfsuugGfuuacaugAfaAfucccasusc 237AD-60636 UfsusUfcAfuGfuAfAfCfcAfaGfaGfuAfuUfL96 206asAfsuAfcUfcUfuGfguuAfcAfuGfaAfasusc 238 AD-68320ususucauGfuAfAfCfcaagaguauuL96 207 asAfsuacUfcUfUfgguuAfcAfugaaasusc 239AD-68326 ususucauGfuAfAfCfcaagaguauuL96 208asAfsuacUfcuugguuAfcAfugaaasusc 240 AD-60611UfsgsUfaAfcCfaAfGfAfgUfaUfuCfcAfuUfL96 209asAfsuGfgAfaUfaCfucuUfgGfuUfaCfasusg 241 AD-68331usgsuaacCfaAfGfAfguauuccauuL96 210 asAfsuggAfaUfAfcucuUfgGfuuacasusg 242AD-68315 usgsuaacCfaAfGfAfguauuccauuL96 211asAfsuggAfauacucuUfgGfuuacasusg 243 AD-68319asasccaaGfaGfUfAfuuccauuuuuL96 212 asAfsaaaUfgGfAfauacUfcUfugguusasc 244AD-60612 AfsasCfcAfaGfaGfUfAfuUfcCfaUfuUfuUfL96 213asAfsaAfaUfgGfaAfuacUfcUfuGfgUfusasc 245 AD-68316asasccaaGfaGfUfAfuuccauuuuuL96 214 asAfsaaaUfggaauacUfcUfugguusasc 246AD-60664 UfsusUfuUfaCfuAfAfAfgCfaGfuGfuUfuUfL96 215asAfsaAfcAfcUfgCfuuuAfgUfaAfaAfasusg 247 AD-68321ususuuuaCfuAfAfAfgcaguguuuuL96 216 asAfsaacAfcUfGfcuuuAfgUfaaaaasusg 248AD-68318 ususuuuaCfuAfAfAfgcaguguuuuL96 217asAfsaacAfcugcuuuAfgUfaaaaasusg 249 AD-60665UfsusAfcUfaAfaGfCfAfgUfgUfuUfuCfaAfL96 218usUfsgAfaAfaCfaCfugcUfuUfaGfuAfasasa 250 AD-60642CfsusAfaAfgCfaGfUfGfuUfuUfcAfcCfuAfL96 219usAfsgGfuGfaAfaAfcacUfgCfuUfuAfgsusa 251 AD-68329csusaaagCfaGfUfGfuuuucaccuaL96 220 usAfsgguGfaaaacacUfgCfuuuagsusa 252AD-68334 csusaaagCfaGfUfGfuuuucaccuaL96 221usAfsgguGfaAfAfacacUfgCfuuuagsusa 253 AD-68328gsgscagaGfaCfAfAfuaaaacauuaL96 222 usAfsaugUfuuuauugUfcUfcugccsusg 254AD-68333 gsgscagaGfaCfAfAfuaaaacauuaL96 223usAfsaugUfuUfUfauugUfcUfcugccsusg 255 AD-60639GfsgsCfaGfaGfaCfAfAfuAfaAfaCfaUfuAfL96 224usAfsaUfgUfuUfuAfuugUfcUfcUfgCfcsusg 256 AD-60643CfsasGfaGfaCfaAfUfAfaAfaCfaUfuCfcUfL96 225asGfsgAfaUfgUfuUfuauUfgUfcUfcUfgscsc 257 AD-68317csasgagaCfaAfUfAfaaacauuccuL96 226 asGfsgaaUfguuuuauUfgUfcucugscsc 258AD-68335 csasgagaCfaAfUfAfaaacauuccuL96 227asGfsgaaUfgUfUfuuauUfgUfcucugscsc 259 AD-68327csasauaaAfaCfAfUfuccugugaaaL96 228 usUfsucaCfaggaaugUfuUfuauugsusc 260AD-68332 csasauaaAfaCfAfUfuccugugaaaL96 229usUfsucaCfaGfGfaaugUfuUfuauugsusc 261 AD-60637CfsasAfuAfaAfaCfAfUfuCfcUfgUfgAfaAfL96 230usUfsuCfaCfaGfgAfaugUfuUfuAfuUfgsusc 262

Example 8. Administration of AD-65492 to Cynomologous Monkeys

The efficacy of AD-65492 was further assessed by administration toCynomologous monkeys.

In a first set of experiments, four Groups (Groups 1, 2, 3, and 7), weresubcutaneously administered a single 0.3 mg/kg dose (Group 1); a singledose of 1 mg/kg (Group 2); a monthly dose of 1 mg/kg for 4 months (QM×4)(Group 7); or a monthly dose of 3 mg/kg for 4 months (QM×4) (Group 3).

Serum, plasma, and sparse liver samples were collected pre-dose and ondays 3, 7, 10, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 91, 98, 112,126, 154, 175, 203, 230, 260, 290, 310, 335, and 364 for Groups 3 and 4.

For Groups 1 and 2, serum was collected on days −7 and −1 pre-dose, anddays 3, 7, 10, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 91, 105, and119 post-dose; plasma was collected on day 1 pre-dose, and 0.5, 1, 2, 4,8, 24, 48, 96 and 168 hours post-dose.

For Groups 3 and 7 serum was collected on days −7 and −1 pre-dose, anddays 3, 7, 10, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 91, 98, 112,127, 155, 176, 204, 232, 260, 288, 316, 344 and 372 post-dose; plasmawas collected on day 1 pre-dose, and 0.5, 1, 2, 4, 8, 12, 24, 48, 96 and168 hours post-dose; plasma was also collected on day 85 pre-dose and0.5, 1, 2, 4, 8, 12, 24, 48, 96 and 168 hours post-dose.

For Group 7, sparse liver samples were collected on day 1, eight hourspre-dose (i.e., 8 hours before the dose of AD-65492 was administered tothe subject, a sparse liver sample was collected from the subject); andon post-dose day 7; day 22; day 29, eight hours pre-dose; day 57, eighthours pre-dose; day 85, eight hours pre-dose; day 91; day 106; and day141.

For Group 3, sparse liver samples were collected post-dose on day 29,eight hours pre-dose; day 57, eight hours pre-dose; day 85, eight hourspre-dose; day 91, day 106, and day 141.

The serum level of TTR protein was determined as described above.

FIG. 11 provides the results of these studies, and shows robustsuppression of TTR expression achieved by administration of AD-65492.The data demonstrate that AD-65492 provides greater than 90% sustainedsuppression of TTR expression for approximately 6 weeks and 17 weekspost-final dose following monthly dosing for four months (QM×4) with 1mg/kg (Group 7) of AD-65492 or 3 mg/kg of AD-65492 (Group 3),respectively. The data also demonstrate about 40% suppression of TTRexpression at 17 weeks post-final dose following monthly dosing for fourmonths (QM×4) with 1 mg/kg of AD-65492 (Group 7).

The data indicate that quarterly dosing of human subjects with AD-65492would be effective in suppressing TTR expression at a dose levelintermediate to 1 mg/kg and 3 mg/kg, e.g., 2 mg/kg, assuming a 1:1translation between dosing in Cynomologous monkeys and humans.

In a second set of experiments, three Groups (Groups 9, 10, and 11; seeFIG. 12), were subcutaneously administered a monthly dose of either 0.3mg/kg for 6 months (QM×6) (Group 9); a monthly dose of 0.6 mg/kg for 6months (QM×6) (Group 10); or an initial dose of 1 mg/kg followed by amonthly maintenance dose of 0.3 mg/kg for one month (QM×1) after theinitial dose for five months (QM×5) (Group 11).

Serum samples were collected on days −7 and −1 pre-dose, and days 4, 8,11, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, 85, 92, 99, 113, 127, 155,176, and 204, 232, 260 and 288. post-dose. The serum level of TTRprotein was determined as described above.

FIG. 13 provides the results of these studies, and demonstrates robustsuppression of TTR achieved by AD-65492 after a monthly dose of 0.6mg/kg for two months (QM×2), which is comparable to suppression of TTRexpression following a 1 mg/kg initial dose. The data also demonstratethat AD-65492 provides about 80% sustained suppression of TTR expressionafter monthly dosing for two months at 0.3 mg/kg; three out of fourmonkeys achieved 60%-85% TTR suppression following a monthly 0.3 mg/kgdoes of AD-65492 for two months. Up to 90% suppression of TTR expressionwas demonstrated following a single 1 mg/kg dose, with a second dose of0.3 mg/kg maintaining nadir for three weeks post dose second dose.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments and methods described herein. Such equivalents are intendedto be encompassed by the scope of the following claims.

We claim:
 1. A method of prophylactically treating a human subject atrisk for developing a TTR-associated disease, comprising administeringto the subject a prophylactically effective amount of a double strandedribonucleic acid (RNAi) agent, wherein the double stranded RNAi agentcomprises a sense strand differing by no more than 4 modifiednucleotides from the nucleotide sequence of5′-usgsggauUfuCfAfUfguaaccaaga-3′ (SEQ ID NO: 10) and an antisensestrand differing by no more than 4 modified nucleotides from thenucleotide sequence 5′-usCfsuugGfuuAfcaugAfaAfucccasusc-3′ (SEQ ID NO:7), wherein a, c, g, and u are 2′-O-methyl (2′-OMe) A, C, G, and U; Af,Cf, Gf, and Uf are 2′-fluoro A, C, G, and U; and s is a phosphorothioatelinkage, thereby prophylactically treating the subject.
 2. The method ofclaim 1, wherein said subject carries a TTR gene mutation that isassociated with the development of a TTR-associated disease.
 3. Themethod of claim 1, wherein said TTR-associated disease is selected fromthe group consisting of senile systemic amyloidosis (SSA), systemicfamilial amyloidosis, familial amyloidotic polyneuropathy (FAP),familial amyloidotic cardiomyopathy (FAC), leptomeningeal/CentralNervous System (CNS) amyloidosis, and hyperthyroxinemia.
 4. The methodof claim 1, wherein said double stranded RNAi agent is administered tosaid subject by an administration means selected from the groupconsisting of subcutaneous, intravenous, intramuscular, intrabronchial,intrapleural, intraperitoneal, intraarterial, lymphatic, cerebrospinal,and any combinations thereof.
 5. The method of claim 1, wherein saiddouble stranded RNAi agent is administered to said subject viasubcutaneous administration.
 6. The method of claim 5, wherein thesubcutaneous administration is self-administration.
 7. The method ofclaim 6, wherein the self-administration is via a pre-filled syringe orauto-injector syringe.
 8. The method of claim 1, wherein the doublestranded RNAi agent is chronically administered to the subject.
 9. Themethod of claim 1, wherein the sense strand differs by no more than 3modified nucleotides from the nucleotide sequence of5′-usgsggauUfuCfAfUfguaaccaaga-3′ (SEQ ID NO: 10) and the antisensestrand differs by no more than 3 modified nucleotides from thenucleotide sequence 5′-usCfsuugGfuuAfcaugAfaAfucccasusc-3′ (SEQ ID NO:7).
 10. The method of claim 1, wherein the sense strand differs by nomore than 2 modified nucleotides from the nucleotide sequence of5′-usgsggauUfuCfAfUfguaaccaaga-3′ (SEQ ID NO: 10) and the antisensestrand differs by no more than 2 modified nucleotides from thenucleotide sequence 5′-usCfsuugGfuuAfcaugAfaAfucccasusc-3′ (SEQ ID NO:7).
 11. The method of claim 1, wherein the sense strand differs by nomore than 1 modified nucleotide from the nucleotide sequence of5′-usgsggauUfuCfAfUfguaaccaaga-3′ (SEQ ID NO: 10) and the antisensestrand differs by no more than 1 modified nucleotide from the nucleotidesequence 5′-usCfsuugGfuuAfcaugAfaAfucccasusc-3′ (SEQ ID NO: 7).
 12. Themethod of claim 1, wherein the sense strand comprises the nucleotidesequence 5′-usgsggauUfuCfAfUfguaaccaaga-3′ (SEQ ID NO: 10) and theantisense strand comprises the nucleotide sequence5′-usCfsuugGfuuAfcaugAfaAfucccasusc-3′ (SEQ ID NO: 7).
 13. The method ofclaim 1, wherein the sense strand consists of the nucleotide sequence5′-usgsggauUfuCfAfUfguaaccaaga-3′ (SEQ ID NO: 10) and the antisensestrand consists of the nucleotide sequence5′-usCfsuugGfuuAfcaugAfaAfucccasusc-3′ (SEQ ID NO: 7).
 14. The method ofany one of claims 1 and 9-13, wherein the double stranded RNAi agentfurther comprises at least one ligand.
 15. The method of any one ofclaims 1 and 9-13, wherein the sense strand of the double stranded RNAiagent is conjugated to at least one ligand.
 16. The method of claim 15,wherein the ligand is one or more GalNAc derivatives attached through abivalent or trivalent branched linker.
 17. The method of claim 15,wherein the ligand is


18. The method of claim 15, wherein the ligand is attached to the 3′ endof the sense strand.
 19. The method of claim 18, wherein the doublestranded RNAi agent is conjugated to the ligand as shown in thefollowing schematic

wherein X is O or S.
 20. The method of claim 19, wherein the X is
 0. 21.A method of prophylactically treating a human subject at risk fordeveloping a TTR-associated disease, comprising administering to thesubject a prophylactically effective amount of a double strandedribonucleic acid (RNAi) agent, wherein the double stranded RNAi agentcomprises a sense strand and an antisense strand, wherein the sensestrand comprises the nucleotide sequence5′-usgsggauUfuCfAfUfguaaccaagaL96-3′ (SEQ ID NO: 10) and the antisensestrand comprises the nucleotide sequence5′-usCfsuugGfuuAfcaugAfaAfucccasusc-3′ (SEQ ID NO: 7) wherein a, c, g,and u are 2′-O-methyl (2′-OMe) A, C, G, and U; Af, Cf, Gf, and Uf are2′-fluoro A, C, G, and U; s is a phosphorothioate linkage; and L96 isN-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol, therebyprophylactically treating the subject.
 22. The method of claim 21,wherein the subject carries a TTR gene mutation that is associated withthe development of a TTR-associated disease.
 23. The method of claim 21,wherein the TTR-associated disease is selected from the group consistingof senile systemic amyloidosis (SSA), systemic familial amyloidosis,familial amyloidotic polyneuropathy (FAP), familial amyloidoticcardiomyopathy (FAC), leptomeningeal/Central Nervous System (CNS)amyloidosis, and hyperthyroxinemia.
 24. The method of claim 21, whereinthe double stranded RNAi agent is administered to said subject by anadministration means selected from the group consisting of subcutaneous,intravenous, intramuscular, intrabronchial, intrapleural,intraperitoneal, intraarterial, lymphatic, cerebrospinal, and anycombinations thereof.
 25. The method of claim 21, wherein the doublestranded RNAi agent is administered to the subject via subcutaneousadministration.
 26. The method of claim 25, wherein the subcutaneousadministration is self-administration.
 27. The method of claim 26,wherein the self-administration is via a pre-filled syringe orauto-injector syringe.
 28. The method of claim 21, wherein the doublestranded RNAi agent is chronically administered to the subject.